Anti calr antibody

1 mg protein extracted from AGS cells was immunoprecipitated (IP) overnight with anti-CALR antibody (Abcam, Shanghai, China), anti-Snail antibody and anti-G9a antibody (Cell Signaling Technology, USA), or rabbit or normal mouse IgG (Beyotime, Jiangsu, China) at 4 °C and then with protein A + G agarose beads (Beyotime, Jiangsu, China) for at least 2 h. After IP, the potential proteins binding with CALR were analyzed by MS analysis (SANGON BIOTECH, Shanghai, China). To verify the results of MS analysis, the beads were then boiled in 30 μL of 1% SDS loading buffer for western blotting with Snail (Cell Signaling Technology, USA), G9a (Proteintech, USA), or CALR antibodies (Sigma-Aldrich, USA).

Following treatments, cells were transferred to a 96-well plate and washed twice with 150 µl/well of cold (4 °C) PBS. Then, they were resuspended in 50 µl/well of anti-CALR antibody (Abcam, Cambridge, UK) diluted in PBS supplemented with 1% bovine serum albumin (BSA) and incubated for 45 min on ice. Afterwards, cells were washed twice with 150 µl/well of cold PBS and resuspended in 50 µl/well of goat anti-rabbit IgG-Alexa Fluor 488 or -Alexa Fluor 647 (Invitrogen) diluted in PBS/1% BSA. After 30 min of incubation on ice and two additional washing steps, cells were resuspended in 120 µl/well of cold PBS supplemented with DAPI at 1 µg/mL [61 (link), 62 (link)]. Cells were analyzed by means of a MACSQuant flow cytometer and data were analyzed with FlowJo software.