Hrp conjugated goat anti mouse igg

Human embryonic kidney 293T (HEK293T) cells were transfected using Lipofectamine 2000 (Thermo Fisher, USA) at a 1:1 ratio with either the WT or mutant plasmid. IFN-γ (10⁵ IU/mL, Peprotech, USA) or IFN-α (10⁵ IU/mL, MCE, USA) was used to stimulate the transfected cells for 30 or 60 minutes, or normal saline was used as a control. Western blot analysis was used to assess GAPDH, total STAT1 protein, and phospho-STAT1 (Y701). The primary antibodies used here included mouse anti-Flag (1:2000, Sigma, F1804), rabbit anti-STAT1 (phospho Y701) (1:1000, Abcam, ab109457), and rabbit anti-GAPDH (1:10000, Abcam, ab181602) antibody. The secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG (1:10000, Jackson, #111-035-003) and HRP conjugated Goat Anti-Mouse IgG (1:5000, Servicebio, GB23301).

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).

Paraffin-embedded and formalin-fixed samples were cut into 5-μM sections, which were then processed for IF staining or IHC staining. First incubation with the CD103 (ABCAM) antibody, followed by HRP conjugated Goat Anti-Mouse IgG (Servicebio). Then, incubation with antibodies against human CD8, CD38 (ABCAM), followed by Cy5 conjugated Goat Anti-mouse IgG, FITC conjugated Goat Anti-Rabbit IgG (Servicebio). Images were acquired with a confocal microscopy (Zeiss LSM 710, Carl Zeiss, Dublin, CA).