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and Immunoblot analysis\nFor immunostaining, cells were seeded at a density of 3–5 × 104 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments.\nTotal proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein (determined by Bio-rad Dc Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [12]. 16.5% tris-tricine SDS-PAGE gels were used for subunit c immunoblotting experiments."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T6623","span":{"begin":59,"end":64},"obj":"CL_GO_EXT:cell"},{"id":"T6624","span":{"begin":103,"end":108},"obj":"CL_GO_EXT:cell"},{"id":"T6625","span":{"begin":147,"end":152},"obj":"GO:0040007"},{"id":"T6626","span":{"begin":230,"end":242},"obj":"CHEBI:16842"},{"id":"T6627","span":{"begin":292,"end":300},"obj":"CHEBI:17790"},{"id":"T6628","span":{"begin":301,"end":308},"obj":"CHEBI:15347"},{"id":"T6629","span":{"begin":363,"end":371},"obj":"GO:0042571"},{"id":"T6630","span":{"begin":384,"end":389},"obj":"CL_GO_EXT:cell"},{"id":"T6631","span":{"begin":518,"end":525},"obj":"CHEBI_SO_EXT:glycine"},{"id":"T6632","span":{"begin":563,"end":571},"obj":"GO:0042571"},{"id":"T6633","span":{"begin":583,"end":595},"obj":"CHEBI:9750"},{"id":"T6634","span":{"begin":641,"end":647},"obj":"NCBITaxon:27592"},{"id":"T6635","span":{"begin":648,"end":653},"obj":"UBERON:0001977"},{"id":"T6636","span":{"begin":648,"end":661},"obj":"PR_EXT:000003918"},{"id":"T6637","span":{"begin":699,"end":707},"obj":"GO:0042571"},{"id":"T6638","span":{"begin":756,"end":764},"obj":"GO:0042571"},{"id":"T6639","span":{"begin":1074,"end":1083},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T6640","span":{"begin":1088,"end":1094},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T6641","span":{"begin":1127,"end":1136},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T6642","span":{"begin":1141,"end":1147},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T6643","span":{"begin":1249,"end":1257},"obj":"CHEBI_PR_EXT:protein"},{"id":"T6644","span":{"begin":1277,"end":1281},"obj":"CL_GO_EXT:cell"},{"id":"T6645","span":{"begin":1324,"end":1328},"obj":"CHEBI:9754"},{"id":"T6646","span":{"begin":1341,"end":1353},"obj":"CHEBI:9750"},{"id":"T6647","span":{"begin":1355,"end":1363},"obj":"GO:0016020"},{"id":"T6648","span":{"begin":1393,"end":1412},"obj":"CHEBI_GO_EXT:peptidase_or_protease_or_proteinase_inhibitor"},{"id":"T6649","span":{"begin":1446,"end":1457},"obj":"CHEBI:7989"},{"id":"T6650","span":{"begin":1486,"end":1495},"obj":"CHEBI:6426"},{"id":"T6651","span":{"begin":1674,"end":1681},"obj":"CHEBI_PR_EXT:protein"},{"id":"T6652","span":{"begin":1708,"end":1715},"obj":"CHEBI_PR_EXT:protein"},{"id":"T6653","span":{"begin":1740,"end":1743},"obj":"CHEBI:8984"},{"id":"T6654","span":{"begin":1806,"end":1810},"obj":"CHEBI:9754"},{"id":"T6655","span":{"begin":1811,"end":1818},"obj":"CHEBI:46760"},{"id":"T6656","span":{"begin":1819,"end":1822},"obj":"CHEBI:8984"},{"id":"T6657","span":{"begin":1847,"end":1856},"obj":"PR_EXT:000022190"}],"text":"Immunostaining and Immunoblot analysis\nFor immunostaining, cells were seeded at a density of 3–5 × 104 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments.\nTotal proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein (determined by Bio-rad Dc Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [12]. 16.5% tris-tricine SDS-PAGE gels were used for subunit c immunoblotting experiments."}

{"project":"2_test","denotations":[{"id":"15588329-12374761-12950169","span":{"begin":1795,"end":1797},"obj":"12374761"}],"text":"Immunostaining and Immunoblot analysis\nFor immunostaining, cells were seeded at a density of 3–5 × 104 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments.\nTotal proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein (determined by Bio-rad Dc Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [12]. 16.5% tris-tricine SDS-PAGE gels were used for subunit c immunoblotting experiments."}