PMC:3245220 / 15646-17455 JSONTXT

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T7333","span":{"begin":0,"end":5},"obj":"P09603"},{"id":"T7334","span":{"begin":2,"end":5},"obj":"P04141"},{"id":"T7335","span":{"begin":14,"end":18},"obj":"P17252"},{"id":"T7336","span":{"begin":132,"end":137},"obj":"P09603"},{"id":"T7337","span":{"begin":134,"end":137},"obj":"P04141"},{"id":"T7338","span":{"begin":219,"end":223},"obj":"P17252"},{"id":"T7339","span":{"begin":251,"end":256},"obj":"P09603"},{"id":"T7340","span":{"begin":253,"end":256},"obj":"P04141"},{"id":"T7341","span":{"begin":272,"end":276},"obj":"P17252"},{"id":"T7342","span":{"begin":691,"end":696},"obj":"P09603"},{"id":"T7343","span":{"begin":693,"end":696},"obj":"P04141"},{"id":"T7344","span":{"begin":849,"end":853},"obj":"P17252"},{"id":"T7345","span":{"begin":889,"end":893},"obj":"P17252"},{"id":"T24373","span":{"begin":977,"end":982},"obj":"P09603"},{"id":"T24374","span":{"begin":979,"end":982},"obj":"P04141"},{"id":"T24375","span":{"begin":993,"end":997},"obj":"P17252"},{"id":"T24376","span":{"begin":1031,"end":1036},"obj":"P09603"},{"id":"T24377","span":{"begin":1033,"end":1036},"obj":"P04141"},{"id":"T24378","span":{"begin":1406,"end":1410},"obj":"P17252"},{"id":"T24379","span":{"begin":1600,"end":1604},"obj":"P17252"},{"id":"T24380","span":{"begin":1652,"end":1657},"obj":"P09603"},{"id":"T24381","span":{"begin":1654,"end":1657},"obj":"P04141"},{"id":"T24382","span":{"begin":1751,"end":1756},"obj":"P09603"},{"id":"T24383","span":{"begin":1753,"end":1756},"obj":"P04141"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

{"project":"bionlp-st-ge-2016-reference","denotations":[{"id":"T7346","span":{"begin":230,"end":247},"obj":"Regulation"},{"id":"T7347","span":{"begin":14,"end":18},"obj":"Protein"},{"id":"T7348","span":{"begin":219,"end":223},"obj":"Protein"},{"id":"T7349","span":{"begin":0,"end":5},"obj":"Protein"},{"id":"T7350","span":{"begin":132,"end":137},"obj":"Protein"},{"id":"T7351","span":{"begin":251,"end":256},"obj":"Protein"},{"id":"T7352","span":{"begin":691,"end":696},"obj":"Protein"},{"id":"T24384","span":{"begin":983,"end":992},"obj":"Positive_regulation"},{"id":"T24385","span":{"begin":993,"end":997},"obj":"Protein"},{"id":"T24386","span":{"begin":1406,"end":1410},"obj":"Protein"},{"id":"T24387","span":{"begin":1600,"end":1604},"obj":"Protein"},{"id":"T24388","span":{"begin":977,"end":982},"obj":"Protein"},{"id":"T24389","span":{"begin":1031,"end":1036},"obj":"Protein"},{"id":"T24390","span":{"begin":1652,"end":1657},"obj":"Protein"},{"id":"T24391","span":{"begin":1751,"end":1756},"obj":"Protein"}],"relations":[{"id":"R5490","pred":"themeOf","subj":"T7348","obj":"T7346"},{"id":"R5491","pred":"causeOf","subj":"T7351","obj":"T7346"},{"id":"R18644","pred":"themeOf","subj":"T24385","obj":"T24384"},{"id":"R18645","pred":"causeOf","subj":"T24388","obj":"T24384"}],"attributes":[{"id":"M28","pred":"Speculation","subj":"T7346","obj":"true"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

{"project":"bionlp-st-ge-2016-reference-tees","denotations":[{"id":"T7418","span":{"begin":14,"end":25},"obj":"Protein"},{"id":"T7419","span":{"begin":78,"end":82},"obj":"Protein"},{"id":"T7420","span":{"begin":111,"end":116},"obj":"Protein"},{"id":"T7421","span":{"begin":132,"end":137},"obj":"Protein"},{"id":"T7422","span":{"begin":214,"end":217},"obj":"Protein"},{"id":"T7423","span":{"begin":219,"end":223},"obj":"Protein"},{"id":"T7424","span":{"begin":251,"end":256},"obj":"Protein"},{"id":"T7425","span":{"begin":100,"end":110},"obj":"Positive_regulation"},{"id":"T7426","span":{"begin":241,"end":247},"obj":"Positive_regulation"},{"id":"T7427","span":{"begin":272,"end":276},"obj":"Protein"},{"id":"T7428","span":{"begin":368,"end":378},"obj":"Protein"},{"id":"T7429","span":{"begin":744,"end":747},"obj":"Protein"},{"id":"T7430","span":{"begin":828,"end":853},"obj":"Protein"},{"id":"T7431","span":{"begin":889,"end":893},"obj":"Protein"},{"id":"T24459","span":{"begin":977,"end":982},"obj":"Protein"},{"id":"T24460","span":{"begin":993,"end":997},"obj":"Protein"},{"id":"T24461","span":{"begin":983,"end":992},"obj":"Positive_regulation"},{"id":"T24462","span":{"begin":1031,"end":1036},"obj":"Protein"},{"id":"T24463","span":{"begin":1117,"end":1134},"obj":"Protein"},{"id":"T24464","span":{"begin":1138,"end":1158},"obj":"Protein"},{"id":"T24465","span":{"begin":1204,"end":1214},"obj":"Protein"},{"id":"T24466","span":{"begin":1406,"end":1410},"obj":"Protein"},{"id":"T24467","span":{"begin":1534,"end":1537},"obj":"Protein"},{"id":"T24468","span":{"begin":1600,"end":1604},"obj":"Protein"},{"id":"T24469","span":{"begin":1652,"end":1657},"obj":"Protein"},{"id":"T24470","span":{"begin":1751,"end":1756},"obj":"Protein"}],"relations":[{"id":"R5506","pred":"themeOf","subj":"T7420","obj":"T7425"},{"id":"R5507","pred":"causeOf","subj":"T7423","obj":"T7426"},{"id":"R5508","pred":"themeOf","subj":"T7424","obj":"T7426"},{"id":"R18658","pred":"causeOf","subj":"T24459","obj":"T24461"},{"id":"R18659","pred":"themeOf","subj":"T24460","obj":"T24461"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

{"project":"events-check-again","denotations":[{"id":"T7411","span":{"begin":0,"end":5},"obj":"Protein"},{"id":"T7412","span":{"begin":14,"end":18},"obj":"Protein"},{"id":"T7413","span":{"begin":132,"end":137},"obj":"Protein"},{"id":"T7414","span":{"begin":219,"end":223},"obj":"Protein"},{"id":"T7415","span":{"begin":230,"end":247},"obj":"Regulation"},{"id":"T7416","span":{"begin":251,"end":256},"obj":"Protein"},{"id":"T7417","span":{"begin":691,"end":696},"obj":"Protein"},{"id":"T24451","span":{"begin":977,"end":982},"obj":"Protein"},{"id":"T24452","span":{"begin":983,"end":992},"obj":"Positive_regulation"},{"id":"T24453","span":{"begin":993,"end":997},"obj":"Protein"},{"id":"T24454","span":{"begin":1031,"end":1036},"obj":"Protein"},{"id":"T24455","span":{"begin":1406,"end":1410},"obj":"Protein"},{"id":"T24456","span":{"begin":1600,"end":1604},"obj":"Protein"},{"id":"T24457","span":{"begin":1652,"end":1657},"obj":"Protein"},{"id":"T24458","span":{"begin":1751,"end":1756},"obj":"Protein"}],"relations":[{"id":"R5504","pred":"themeOf","subj":"T7414","obj":"T7415"},{"id":"R5505","pred":"causeOf","subj":"T7416","obj":"T7415"},{"id":"R18656","pred":"causeOf","subj":"T24451","obj":"T24452"},{"id":"R18657","pred":"themeOf","subj":"T24453","obj":"T24452"}],"attributes":[{"id":"M34","pred":"Speculation","subj":"T7415","obj":"true"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

{"project":"GO-CC","denotations":[{"id":"T7368","span":{"begin":593,"end":598},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T7369","span":{"begin":732,"end":742},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T7370","span":{"begin":828,"end":836},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T7371","span":{"begin":732,"end":742},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T7372","span":{"begin":828,"end":836},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T24405","span":{"begin":1126,"end":1134},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T24406","span":{"begin":1150,"end":1158},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T24407","span":{"begin":1126,"end":1134},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T24408","span":{"begin":1150,"end":1158},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T24409","span":{"begin":1146,"end":1158},"obj":"http://purl.obolibrary.org/obo/GO_0071736"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

{"project":"GO-MF","denotations":[{"id":"T7359","span":{"begin":14,"end":18},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T7360","span":{"begin":219,"end":223},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T7361","span":{"begin":272,"end":276},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T7362","span":{"begin":849,"end":853},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T7363","span":{"begin":214,"end":217},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T7364","span":{"begin":368,"end":371},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T7365","span":{"begin":19,"end":34},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T7366","span":{"begin":732,"end":742},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T7367","span":{"begin":828,"end":836},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T24398","span":{"begin":993,"end":997},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T24399","span":{"begin":1406,"end":1410},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T24400","span":{"begin":1122,"end":1125},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T24401","span":{"begin":1204,"end":1207},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T24402","span":{"begin":1126,"end":1134},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T24403","span":{"begin":1150,"end":1158},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T24404","span":{"begin":1208,"end":1223},"obj":"http://purl.obolibrary.org/obo/GO_0016301"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

{"project":"GO-BP","denotations":[{"id":"T7432","span":{"begin":120,"end":137},"obj":"http://purl.obolibrary.org/obo/GO_0036005"},{"id":"T7433","span":{"begin":120,"end":137},"obj":"http://purl.obolibrary.org/obo/GO_1903970"},{"id":"T7434","span":{"begin":120,"end":137},"obj":"http://purl.obolibrary.org/obo/GO_1903971"},{"id":"T7435","span":{"begin":19,"end":34},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T7436","span":{"begin":100,"end":116},"obj":"http://purl.obolibrary.org/obo/GO_0051092"},{"id":"T7437","span":{"begin":120,"end":137},"obj":"http://purl.obolibrary.org/obo/GO_1903969"},{"id":"T24471","span":{"begin":977,"end":992},"obj":"http://purl.obolibrary.org/obo/GO_1901258"},{"id":"T24472","span":{"begin":977,"end":992},"obj":"http://purl.obolibrary.org/obo/GO_1903971"},{"id":"T24473","span":{"begin":1208,"end":1223},"obj":"http://purl.obolibrary.org/obo/GO_0016301"}],"text":"M-CSF-Induces PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

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PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

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PKCα Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-κB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCα was a downstream target of M-CSF in human MDMs. PKCα was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCα demonstrated that equal amounts of PKCα were assayed (Figure 4, middle panel).\n10.1371/journal.pone.0028081.g004 Figure 4 M-CSF activates PKCα in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCα were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCα activity in non-stimulated samples compared to M-CSF-treated MDM ± S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were ≤0.05.\n\nM"}

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