Gpr41

Tissues embedded in OCT compounds were cut into 5-μm sections. The sections were fixed with cold acetone for 10 min at 4 °C and then blocked with 5% BSA for 1 h. Samples were then incubated overnight with the indicated primary antibodies: Muc2 (1:500, Proteintech), GPR41 (1:400, Abcam) and GPR43 (1:400, Abcam) , CA1(1:200, Proteintech), Lgr5 (1:400, Affinity Biosciences), CHGA (1:100, Abcam), RhoB (1:200, Santa Cruz, CA, USA). Samples were washed with PBS and incubated for 2 h with Alexa Fluor 488-labeled second antibody or Alexa Fluor 594-labeled second antibody (1:500, Proteintech). Following extensive washing, the sections were mounted with DAPI Fluoromount-G gum. Images of sections were obtained using a confocal fluorescence microscope (Leica TCS-SP8, Leica Microsystems, Germany).
SW480 cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.25% TritonX-100 for 10 min. The cells were blocked with 5% normal goat serum for 1 h, and incubated overnight with primary antibodies against Muc2 (1:500, Proteintech) and Ki67 (1:250, Abcam), followed by a washing step and incubation with Alexa Fluor 488-labeled second antibody and DAPI. Fluorescence was detected using a fluorescence microscope (OLYMPUS IX73, OLYMPUS, Japan).

The protein expression of CAV-1, ACE2 and GPR41 were detected by western blot. The RIPA lysis buffer (Solarbio) was used to extract total protein from cardiovascular tissue. The extracted protein was separated by 10% SDS-PAGE gel electrophoresis, then transferred to a PVDF membrane, blocked PVDM with 5% skimmed milk powder for 1 hour, and then incubated with CAV-1 (Sigma-Aldrich, Shanghai, China), ACE2 (Proteintech, Wuhan, China) and GPR41 (Abcam, Shanghai, China) antibodies at 4 ° C overnight. The next day, the PVDF membrane was cleaned three times with TBST (Solarbio) for 5 minutes each time. After incubating the correspondding secondary antibody at room temperature for 2 hours, the PVDF membrane was cleaned three times with TBST (Solarbio) for 5 minutes each time again. Observing protein bands using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.).