Anti bak

Proteins were isolated from stably transfected cells at 90% confluence using the RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China). Their concentrations were quantified using a BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China), and the proteins (30 µg/lane) were separated via SDS-PAGE before transfer to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Following this, the membrane with the proteins was subjected to the following incubations and washes: 5% lipid-free milk solution at 37°C for 1.5 h, primary antibody at 4°C overnight (ON), HRP-conjugated secondary antibody (1:1,000; Abcam, Shanghai, China) for 2 h at 37°C, and three TBST buffer washes. Finally, the protein signals were visualized using an enhanced chemiluminescence detection system with a chemiluminescent HRP substrate (Millipore, MA, USA). The antibodies used for protein detection were as follows: anti-UQCRC2 (1:1000, Abcam, Shanghai, China), anti-GAPDH (1:5000; Cell Signaling Technology, MA, USA), anti-cleaved caspase-3 (1:200, Cell Signaling Technology, MA, USA), anti-Bcl-2 (1:1000, Abcam, Shanghai, China), anti-Bax (1:1000, Abcam, Shanghai, China), and anti-Bak (1:10,000, Abcam, Shanghai, China).

Protein extraction kit (Solarbio, China) was used to isolate proteins, and then the concentration of the proteins was detected by BCA Protein Assay kit (Cwbio, China). The cell lysate was loaded on 10% SDS-PAGE after being boiled for 5 min to denaturation and then transferred onto a PVDF membrane (Millipore, USA). The non-specific antigens were blocked using 5% skimmed milk. Primary antibodies were incubated at 4 °C overnight. After removing the appropriate horseradish peroxidase-conjugated secondary antibody (ab97051, 1:3000, Abcam, USA), the blots were detected using Enhanced Chemiluminescence Detection kit (GE Healthcare Biosciences). The primary antibodies used in the experiment were as follows: N-cadherin (ab18203, 1:1000), Vimentin (ab137321, 1:1500), anti-PAX8 (ab53940,1:200), anti-p-Erk1/2 (ab201015, 1:1000), anti-Bcl-2 (ab593480, 1:700), anti-Bax (ab53154, 1:1000), anti-Bak (ab32371, 1:10,000) purchased from Abcam. E-cadherin (3195, 1:1000), anti-p-Akt (Thr308) (13,038, 1:1000), p-JNK1/2(4668, 1:2000), anti-GAPDH (5174, 1:1000), anti-β-actin (4970, 1:1000) purchased from Cell Signaling Technology.