Ap3434a

Cell protein was collected by RIPA lysis buffer (KeyGEN, Nanjing, China) containing 1 nM PMSF (Biotool, Houston, TX, USA). 20 μg protein per well was electrophorsed in 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and incubated with different primary antibodies, including GALNT3 (16716–1-AP, Proteintech, China), caspase3 (ab13847, Abcam, the UK), cleaved caspase3 (ab13585, Abcam, the UK), PARP (ab74290, Abcam, the UK), cleaved PARP (ab4830, Abcam, the UK), MUC1 (ab15481, Abcam, the UK), PI3K-p110α (21890–1-AP, Proteintech, China), p-AKT 308 (AP3743a, Abgent, China), p-AKT 473 (AP3434a, Abgent, China), AKT (ab8805, Abcam, the UK), NF-κB (AP50006, Abgent, China) and GAPDH (AP7873a, Abgent, China), at 4 °C overnight. The membrane was treated with anti-rabbit IgG at 37 °C for 2 h. All bands were detected by an ECL Western blot kit (Thermo Fisher Scientific, USA) and analyzed by Lab Works (TM ver4.6, UVP, Bio Imaging Systems, NY, USA). GAPDH was used as control.

Western blotting was performed in accordance with a previously established protocol [33 (link)]. The protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated by 10–15% SDS-PAGE and then transferred to PVDF membranes (GE Healthcare, Shanghai, China) using a wet Trans–Bolt system. After blocking with 5% BSA (Beyotime Biotechnology), the membranes were incubated with specific primary antibodies at 4 °C, including anti-AKT (10176-2-AP, Proteintech, Wuhan, China), anti-phospho-AKT (AP3434A, ABGENT, San Diego, USA), anti-muscle-specific ring finger protein 1 (MP3401, ECM bioscience), and anti-glyceraldehyde-3-phosphate dehydrogenase (10494-1-AP, Proteintech). The corresponding secondary antibody conjugated horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA) was then applied to the membrane for 1 h at room temperature. For signal detection, both the enhanced chemiluminescence reagent (ECL, Beyotime Biotechnology) and ChemiScope Capture and Analysis system (ChemiScope 6000, CLiNX, Shanghai, China) were used.