Cell lysis, SDS-page and Western blotting were performed according to standard procedures as described previously [45 (link)]. The following antibodies were used: anti-Beclin-1 (Santa Cruz Biotechnology, Heidelberg, Germany, No. sc-48381), anti-Atg12 (Cell Signaling Technology, Danvers, MA, USA, No. 2010), anti-Atg7 (Abcam, Cambridge, USA, No. ab53255), anti-LC3b (Cell Signaling Technology, No. 3868), anti-p62 (Becton Dickinson, Heidelberg, Germany, No. 610832), anti-Tubulin (Sigma, St. Louis, MO, USA, No. T8203-25UL), anti-Lamin B1 (Abcam, No. ab16048), anti-cleaved PARP (Cell Signaling Technology, No. 5625), anti-CyclinD1 (Cell Signaling Technology, No. 2926) and anti-pH2AX (Cell Signaling Technology, No. 9718).
In order to quantify protein expression, we used ImageJ® (by Wayne Rasband at NIH, Bethesda, MD, USA) for densitometric analysis as described previously [49 (link)]. In brief, band density was measured relative to the untreated control and then adjusted to tubulin as loading control. For analysis of isolated cytosolic and nuclear fractions, tubulin and Lamin B1 were utilized as loading controls and to demonstrate proper separation.
In order to quantify protein expression, we used ImageJ® (by Wayne Rasband at NIH, Bethesda, MD, USA) for densitometric analysis as described previously [49 (link)]. In brief, band density was measured relative to the untreated control and then adjusted to tubulin as loading control. For analysis of isolated cytosolic and nuclear fractions, tubulin and Lamin B1 were utilized as loading controls and to demonstrate proper separation.
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