Irdye 800cw goat anti mouse igg h l secondary antibody

All reagents and siRNAs were purchased from Santa Cruz Biotechnology, unless otherwise stated. The following reagents from other suppliers were used: phospho-p53 kinase (Ser¹⁵) rabbit mAb (CST, 9284, USA), p53 kinase rabbit mAb (CST, 9282, USA), phospho-p38 kinase (Thr¹⁸⁰/Tyr¹⁸²) rabbit mAb (CST, 4511, USA), p38 kinase rabbit mAb (CST, 8690, USA), phospho-JNK kinase (Thr¹⁸³/Tyr¹⁸⁵) rabbit mAb (CST, 4668, USA), JNK kinase rabbit mAb (CST, 9252, USA), phospho-ERK kinase (Thr²⁰²/Tyr²⁰⁴) rabbit mAb (CST, 4370, USA), ERK kinase rabbit mAb (CST, 4695, USA), α-enolase kinase rabbit mAb (CST, 3810, USA), stathmin kinase rabbit mAb (CST, 3352, USA), phospho-AMP-activated protein kinase (AMPK; Thr¹⁷²) rabbit mAb (CST, 2531, USA), AMPK rabbit mAb (CST, 2532, USA), anti-β-of actin mouse monoclonal IgG1 (Santa Cruz, sc-47778, USA), rabbit anti-mouse IgG (H +L) secondary antibody TRITC (Invitrogen, PA1-28565, USA), IRDye 800CW goat anti-mouse IgG (H + L) secondary antibody (Li-COR, 926-32210, USA), IRDye 800CW goat anti-rabbit IgG (H + L) secondary antibody (Li-COR, 926–32211, USA), Compound C (Calbiochem, 171260, USA), SB203580 (CST, 5633, USA), SP600125 (CST, 8177, USA), U0126 (CST, 9903, USA), antibody against LC3-II (CST, 2775, USA).

The cells were rinsed with PBS three times and lysed in RIPA buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 1 mM PMSF, and 25 mM MgCl₂). The proteins were mixed with 2x Laemmli sample loading buffer (Bio-Rad, USA) and denatured at 95°C for 10 min, then separated using 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, USA). After blocking with 5% nonfat milk in TBST (0.1% Tween-20), the membranes were incubated in primary antibody for BKCa (ab9276, 1 : 1000, Abcam, Cambridge, UK) or PCNA (ab29, 1 : 1000, Abcam, Cambridge, UK) at 4°C overnight followed by incubation with IRDye 800CW Goat anti-Mouse IgG (H + L) secondary antibody (926-32210, LI-COR, USA) for two hours. The bands were developed by an Odyssey® infrared scanner (LI-COR, Lincoln, USA). The protein expression levels were normalized as the ratio of band intensity to β-actin (ab6276, 1 : 5000, Abcam, Cambridge, UK).

CBP rabbit monoclonal antibody (Cell Signaling Technology, USA) and GAPDH mouse monoclonal antibody (Santa Cruz Biotechnology, USA) were used as the primary antibodies; IRDye800CW goat anti-rabbit IgG (H+L) secondary antibody (Li-Cor, USA) and IRDye800CW goat anti-mouse IgG (H+L) secondary antibody (Li-Cor) were used as secondary antibodies for Western blotting.