Gapdh antibody

MDA-MB-231 and MDA-MB-468 cells were lysed and proteins were extracted using high-efficiency RIPA tissue/cell lysis buffer (Solarbio, Beijing, China). Antibodies used were as follows: KCNQ4 antibody (1:1000, Abways, Shanghai, China), PI3K antibody (1:1000, Abways, Shanghai, China), AKT1/2/3 antibody (1:1000, Abways, Shanghai, China), p-AKT antibody (1:1000, Abways, Shanghai, China) and GAPDH antibody (1:5000, CUSABIO, Wuhan, China). Exposure was performed using a chemiluminescence gel imaging system.

The sample from immunoprecipitation was resuspended with 40 ul Elution Buffer, incubated at 70°C for 20 min at 1,400 rpm, and then centrifuged at 13,200 x g for a short time. The supernatant was transferred to a new EP tube on a magnetic separator. Next, complexes were eluted in boiling water for 10 min with 1X SDS sample buffer and separated on 10% SDS-PAGE. With TBST buffer (20 mM Tris-buffered saline and 0.1% Tween-20) containing 5% non-fat milk powder for 1 h at room temperature, the Membrane was incubated with primary antibody: CKAP4 antibody (1:1,000, 16686-1-AP, Proteintech), GAPDH antibody (1:2000, CUSABIO, Wuhan, China) and then with HRP-conjugated secondary antibody. Bound secondary antibody (anti-mouse or anti-rabbit 1:10,000) (Abcam, Cambridge, MA, USA) was detected using the enhanced chemiluminescence (ECL) reagent (170506, Bio-Rad, Hercules, CA, USA).