Hrp linked goat anti mouse and anti rabbit antibodies

Cells were removed from culture dishes using 0.15% trypsin solution or a cell scraper. Next, cells were centrifuged (1000 rcf, 4 min, +4 °C), washed with PBS solution, centrifuged again (1000 rcf, 4 min, +4 °C), and the pellet was lysed in Radioimmunoprecipitation assay buffer (RIPA buffer) containing 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 0.5% SDS, 0.5% sodium deoxycholate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF) and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland) for 15 min on ice. After centrifugation (15,000 rcf, 15 min, +4 °C), a part of the supernatant was taken for protein concentration assay, and another part was used for western blot analysis, as previously described [30 (link)]. The following primary antibodies were used for western blot: anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118), anti-Bcl-xL (#2764), anti-Bax (#2772), anti-Bim (#2933), anti-Mcl-1 (#5453), anti-Bak (#6947), anti-rabbit full and cleaved caspase-3 (#9662) antibodies (Cell Signaling Technology, Danvers, MA, USA); anti-Bcl-2 (sc-509) (Santa Cruz Biotechnology, Dallas, TX, USA); anti- Poly(ADP Ribose) Polymerase (PARP) (#137653) and anti-tubulin-α (#7291) antibodies (Abcam, Cambridge, UK). HRP-linked goat anti-mouse and anti-rabbit antibodies (#97046 and #97200, respectively; Abcam) were used as secondary antibodies.

After the indicated time of treatment, cells were removed from culture dishes using a cell scraper. Next, cells were centrifuged (500 rcf, 5 min, 4 °C) and washed with ice-cold PBS (Paneco). Then, the cell pellet was lysed in RIPA buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, cOmplete™ Protease Inhibitor Cocktail (Roche)) for 20 min on ice. Lysates were centrifuged (16,000 rcf, 20 min, 4 °C), and a part of the supernatant was taken for the protein concentration assay, and another part was used for Western Blot analysis, as previously described [14 (link)]. The densitometric analysis was performed using Image Lab Software or ImageJ 4.1.
The following primary antibodies were used for WB: anti-GAPDH (#2118), anti-p21 (#2947), anti-Puma (#4976) (all from Cell Signaling Technology), anti-PARP (#137653) (Abcam) and anti-p53 (012M4795) (Sigma) antibodies. HRP-linked goat anti-mouse and anti-rabbit antibodies (#97046 and #97200, respectively; both from Abcam) were used as secondary antibodies.