Anti ryr2

N,N,N′,N′ -Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) and thapsigargin (TG) were obtained from Sigma (St. Louis, MO, United States). BAPTA-AM, EGTA-AM, H89, 2APB, and stattic were purchased from MCE (NJ, United States). KN-93 and KN-92 were obtained from Selleck (Houston, TX, United States). Antibodies including anti-p-STAT3, -STAT3, -p-CaMKII, -CaMKII, -GAPDH, and the secondary antibody were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-IP3R,-p-RyR2 and -SERCA2 were purchased from Abcam (Cambridge, United Kingdom). Anti-RyR2 was purchased from Proteintech Group (Chicago, IL, United States). Anti-ZIP9 was obtained from Biorbyt (Cambridge, United Kingdom). Fluorescence dyes were obtained from Invitrogen (Carlsbad, CA, United States).

The procedures of these two assays have been described earlier [12 (link)]. Anti‐RYR2 (Proteintech) was diluted 1 : 100 to stain CRC TMA slides. Anti‐phospho‐CREB(Ser133) (Affinity, Changzhou, China) was diluted 1 : 100 in an immunofluorescence (IF) assay. Briefly, for immunohistochemistry (IHC), sections of clinical specimens were deparaffinized with xylene and rehydrated with ethanol, followed by staining with anti‐RYR2 antibody and horseradish peroxidase (HRP)‐linked anti‐rabbit IgG, and further developed with 3,3′‐diaminobenzidine (DAB). TMA sections were further photographed and analyzed by Vecture 2 (PerkinElmer). The same algorithm was used to score every core. For IF, cells on slides were fixed in 4% formaldehyde, followed by staining with indicated primary antibodies and fluorescent secondary antibody (Alexa Fluor 488‐conjugated donkey anti‐rabbit IgG and Alexa Fluor 555‐conjugated goat anti‐rabbit IgG, 1 : 1000) and were photographed with a confocal microscope (Zeiss, Cambridge, UK; LSM 880NLO FILM).

The heart total proteins were extracted and determined (KeyGEN BioTECH, Nanjing, China). After boiling, the samples containing 50 µg of proteins were separated using SDS–PAGE and wet-transferred onto PVDF membranes. The PVDF membranes were blocked for 2 h at room temperature with 5% nonfat milk, then incubated with the primary anti-RYR2 (1:800, proteintech, China), anti-HO-1 (1:600, proteintech, China), anti-IL-18 (1:500, abcam, USA) and anti-GAPDH antibodies (1:10000, abcam, USA) overnight at 4 °C. Then, incubating with a secondary HRP-conjugated antibody (1:5000, proteintech, China) was carried out for 2 h at room temperature. GAPDH was used as loading control.