Ab22744

Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes, washed three times with PBS, and then permeabilized in 0.2% Triton X-100 in PBS for 5 minutes. After washing three times with PBS and blocking with PBS containing 5% fetal bovine serum (FBS), the cells were immunostained with rabbit anti-flag (2368s, Cell Signaling), rabbit anti-giantin (ab80864, Abcam), mouse anti-pan-Cadherin (ab22744, Abcam), or mouse anti-atp2c1 (WH0027032M1, Sigma) in PBS with 1% FBS at 4°C overnight. The cells were then washed three times with PBS and incubated with mouse anti-calnexin Alexa Fluor 647 (ab202572, Abcam), goat anti-rabbit Alexa Fluor 488 (A11008, Molecular Probes), donkey anti-mouse Alexa Fluor 488 (ab150105, Abcam), donkey anti-rabbit Alexa Fluor 555 (A31572, Molecular probes), and/or goat anti-rabbit Alexa Fluor 350 (A0408, Beyotime) in PBS with 1% FBS at 37°C for 1.5 hours. The slides were mounted under coverslips using ProLong Gold Antifade reagent (Thermo Fisher Scientific), and fluorescence images were obtained using an LSM-710 microscope (Zeiss, Germany) equipped with a 63X/1.4 numerical aperture oil-immersion objective.

143B and Hela cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). 143B and Hela cells were harvested and then resuspended in cold PBS. The collected cells were suspended in a hypotonic lysing buffer containing PMSF (Beyotime, China) and membrane protein extraction reagent. The resuspended 143B or Hela cells were incubated in ice bath for 15 min and then broken repeatedly using a freeze-thaw method. After centrifugation at 800 × g for 10 min, the supernatant was further centrifuged at 15, 000 × g for 30 min to obtain the cell membrane fragments. The membrane products of CCM-143B and CCM-Hela were lyophilized and stored at −80 °C. The lyophilized membranes are rehydrated in ultrapure water or PBS buffer. For CCM protein characterization, the following antibodies were used for western blot analysis: Anti-pan cadherin antibody (Abcam, ab22744, 1:1000); Anti-Na+/K+-ATPase antibody (Abcam, ab76020, 1:1000), Anti-gp100 antibody (Abcam, ab137078, 1:1000), Anti-Histone H3.3 Rabbit pAb (Servicebio, GB11026, 1:500), Anti-COX IV Rabbit pAb (Servicebio, GB11250, 1:500), Anti-GAPDH Mouse mAb (Servicebio, GB12002, 1:1000), HRP conjugated Goat Anti-Rabbit IgG (Servicebio, GB23303, 1:3000), HRP conjugated Goat Anti-Mouse IgG (Servicebio, GB23301, 1:3000).