Phospho erk

Briefly, polyacrylamide gel electrophoresis was used to separate proteins which extracted from cells, and transferred onto polyvinylidene fluoride membranes. Membranes were incubated with primary antibodies including: FLAG (1:2000; Sigma), E-cadherin (1:500; Santa Cruz, Dallas, TX, USA), N-cadherin (1:500; Santa Cruz), Vimentin (1:1000; Cell Signalling Technology, Beverly, MA, USA), Snail (1:1000; Cell Signalling Technology), JNK (1:1000; Cell Signalling Technology), phospho-JNK (1:500; Cell Signalling Technology), p38 (1:1000; Cell Signalling Technology), phospho-p38 (1:500; Cell Signalling Technology), AKT (1:1000; Cell Signalling Technology), phospho-AKT (1:500; Cell Signalling Technology), FAK (1:1000; Cell Signalling Technology), phospho-FAK (1:500; Cell Signalling Technology), ERK (1:1000; ABclonal Biotech Co., Ltd., Cambridge, MA, USA), phospho-ERK (1:500; Abclonal), IP3R (1:1000; Santa Cruz), STIM1 (1:500; Santa Cruz), STIM2 (1:500; Santa Cruz), SERCA2 (1:500; Santa Cruz), ORAI1 (1:500; Santa Cruz), ORAI2 (1:500; Santa Cruz), ORAI3 (1:500; Santa Cruz), β-actin (1:3000; Proteintech) and then with HRP-conjugated secondary antibody. At last, an enhanced chemiluminescence assay was used to detect the reactions. All original western blots were provided as supplementary materials.

The A431 and A549 cells were cultured in 6-well plates (2×10⁵ cells/well) for 2 h and treated with different concentrations of B7(1, 5, and 10 μM) or 4i (10 μM). The corresponding cells were collected, washed with PBS (P1020, Solarbio), and lysed with RIPA buffer (R0010, Solarbio) to extract the total proteins. The extracted protein was loaded, subjected to SDS-PAGE electrophoresis (Bio-Rad) (Xu et al., 2023 (link)), and then the protein was transferred to a PDVF membrane (IPVH00010, Millipore) and incubated in the corresponding Primary Antibody AKT (9272, Cell Signaling Technology), phospho-AKT (4058, Cell Signaling Technology), ERK (A4782, ABclonal Technology), phospho-ERK (AP0974, ABclonal Technology) and GAPDH (AB0037, Abways Technology) overnight. Then, the Primary Antibody was recovered and enzyme-labeled secondary antibodies Goat Anti-Rabbit IgG HRP (H + L) (A0208, Beyotime Technology) were used. Finally, Imaging was performed on a gel Imaging System (Bio-Rad, United States) using the Ultra-sensitive ECL Chemiluminescence Assay Kit (P0018AS, Beyotime Technology).