Ab194676

Anti-IL-1β antibody (ab106015), anti-caspase-4 antibody (ab25898), anti-caspase-5 antibody (ab40887), anti-GSDMD antibody -C-terminal (ab228824), and anti-Urotensin II antibody-C-terminal (ab194676) were purchased from Abcam (UK, London, Abcam) used for western blot, IF and IHC methods. Anti-caspase-1 antibody (D7F10) was obtained from Cell Signaling Technology (US, Trask Lane Danvers, CST) and was used in western blot, IF, and IHC. Anti-NLRP -3 antibody (Beijing, China, Wanleibio) was used in IHC and IF, and anti-NLRP-3 antibody (ab91413) was obtained from Abcam for western blot.

Gastrocnemius muscle protein was extracted and mixed with 5× loading buffer (Applygen, Beijing, China). The protein was then denatured at 100 ° C for 5 min and then loaded on a SDS-PAGE gel prior to being transferred to NC membranes. Following the immersion of membranes with 5% BSA (AMRESCO Inc., OH, USA), membranes were incubated with primary rabbit polyclonal anti-UII antibody (ab194676, Abcam), primary rabbit polyclonal anti-LC3 antibody (ab48394, Abcam), primary mouse monoclonal anti-p62 antibody (ab56416, Abcam), and primary rabbit polyclonal anti-FNDC5 antibody (ab131390, Abcam) overnight at 4 ° C. Membranes were then incubated with fluorescence-conjugated antirabbit and antimouse antibodies (LI-COR, NE, USA). Semi-quantitative gray-scale intensity was measured by image J. The antibody of GAPDH was purchased from Byeotime (Shanghai, China).

Immunohistochemical analysis was performed on gastrocnemius muscle tissues. The thickness of 5 μm sections were made from the paraffin's embedded muscles. Then 3% hydrogen peroxide was incubated in order to delete endogenous peroxidase. The sections were performed with primary antibodies at 4 ° C overnight, and PBS served as a substitute for the primary antibody in negative control group. The tissue slices were then incubated with second antibody for 30 min.
3,3′-Diaminobenzidine staining was used to distinguish positive antigen from negative antigen. Brown deposits indicated positive staining. Then, 200× high-power microscope fields were randomly selected to calculate the integral optical density of positive staining. The antibodies to UII (ab194676), FNDC5 (ab181884), LC3 (ab48394), and p62 (ab56416) were bought from Abcam (Cambridge, UK).