PMC:4157140 / 35599-37729
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4157140","sourcedb":"PMC","sourceid":"4157140","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4157140","text":"Figure 4 Functional Studies of APOPT1\n(A) Immunoblot analysis of fibroblasts stably expressing APOPT1-HA. No HA-immunoreactive material, visualized using a specific anti-HA antibody (α-HA, Roche), is present in naive conditions, whereas two HA-immunoreactive bands are detected under exposure of the cells to the proteasome inhibitor MG-132. Tubulin and SDHB, immunovisualized by specific antibodies (α-TUB, Sigma-Aldrich; α-SDH30, Mitoscience), are used as loading controls.\n(B) The anti-HA immunoreactive bands obtained as in (A) (arrows) have electrophoretic mobility identical to the in vitro translated cDNAs corresponding to the predicted precursor (prec) and mature (mat) APOPT1-HA protein species, synthesized using the TNT Transcription-Translation System kit (Promega). The in vitro translated products are specific, as no anti-HA immunoreactive material is visualized in the prereaction reticulocyte lysate (ret). Note that the panels are from the same filter, but different exposure times were used to better visualize the bands.\n(C) Anti-HA immunoreactive bands corresponding to the precursor and mature APOPT1 species are detected in fibroblasts exposed to H2O2. Note that the upper band, corresponding to the precursor APOPT1-HA species, is present in the sample collected 8 hr after the exposure to H2O2, whereas only the mature species is detected in samples collected at 24 and 48 hr, suggesting that over time the precursor APOPT1-HA species has been translocated across the inner mitochondrial membrane and quantitatively processed into the mature species by cleavage of a 39 amino acid N-terminal MTS.\n(D) ROS detection by dichlorofluorescein (DCHF, Invitrogen) fluorescence on control (Ct) and individual S2 fibroblasts in basal and oxidative stress conditions. Note that naive individual S2 fibroblasts show significantly higher levels of DCHF fluorescence under exposure to 100 μM and 1 mM H2O2. The levels of DCHF fluorescence are significantly lower in APOPT1-HA-expressing individual S2 fibroblasts. Bars represent standard deviations. The p values were obtained by unpaired, two-tail Student’s t test.","divisions":[{"label":"label","span":{"begin":0,"end":8}},{"label":"p","span":{"begin":10,"end":38}},{"label":"p","span":{"begin":39,"end":476}},{"label":"p","span":{"begin":477,"end":1042}},{"label":"p","span":{"begin":1043,"end":1623}}],"tracks":[]}