Bs 1519r

The total proteins were extracted from the cultured cells in the RIPA buffer containing protease inhibitors. After all the cell lysates were separated by SDS-PAGE, they were transferred to the polyvinylidene fluoride membrane (PVDF). The PVDF membrane was blocked by 5% skim milk at room temperature for 1 h. The membranes were then incubated overnight with the primary antibody at 4°C. The primary antibody included rabbit anti-Gimap5 (14108S, Cell Signaling Technology, Boston, USA, 1:1000), rabbit anti-E-cadherin (bs-1519R, BIOSS, Beijing, China, 1:1000), rabbit anti-N-cadherin (bs-20623R, BIOSS, Beijing, China, 1:1000), rabbit anti-CD-M6PR (BS8108, Bioworlde, Beijing, China, 1:1000), rabbit anti-CI-M6PR (WL02758, Wanleibio, Shenyang, China, 1:500), rabbit anti-PADI4 (BS7314, Bioworlde, Beijing, China, 1:1000) and rabbit anti-GAPDH (BIOSS, Beijing, China, 1:5000). After each membrane was washed with TBST (containing 1% Tween 20) six times for 5 min, all the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (BIOSS, Beijing, China, 1:5000) at room temperature for 1 h. The membranes were eventually visualized using a Tanon 5200 chemiluminescence image analysis system (Tanon, Shanghai, China).

For protein characterization by SDS PAGE, all samples were prepared in loading buffer at a final protein concentration of 1 mg mL⁻¹, as measured by BCA assay (Pierce BCA Protein Assay Kit, Thermo Scientific). The samples were heated at 95 °C for 10 min to fully denature the protein. Then membrane protein was separated using 10% SDS‐PAGE, stained and eluted with Coomassie brilliant blue, and imaged with a ChemiDoc XRS+ System (Bio‐Rad). For WB analysis, the proteins on SDS‐PAGE were transferred to PVDF membrane with transfer buffer (Invitrogen, CA, USA), and stained with antibodies against EpCAM (abs‐120056, absin), antibodies against E‐cadherin (bs‐1519R, Bioss), antibodies against ATPase Na⁺/K⁺ beta 2 (bs‐23413R, Bioss), CD47 antibody (bs‐2386R, Bioss), and HRP‐labeled goat anti‐rabbit IgG (bs‐40295G‐HRP, Bioss) as the secondary antibodies.