Tnf α bsm 33207 m

Western blotting was performed following a previously described procedure with some modifications [25 (link)]. The collected TOCs were lysed using RIPA lysis buffer containing PMSF (Meilunbio, Dalian, China). After incubating on ice for 30 min, the lysate was centrifuged at 10 000 × g for 30 min at 4 °C. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime). Equal amounts of proteins (40 µg) were subjected to 8–12% SDS‒PAGE, and the separated proteins were subsequently transferred to PVDF membranes, after which the proteins were blocked with a closure solution (5% skim milk powder) for 2 h at room temperature. The following primary antibodies were used: IκBα (10268-1-AP; Proteintech), p-IκBα (bs-2513R; Bioss), p65 (bs-0465R; Bioss), p-p65 (bs-0982R; Bioss), TNF-α (bsm-33207 M; Bioss), MLCK (ab232949; Abcam), MLC2 (3672; Cell Signaling Technology), P-MLC2 (3672; Cell Signaling Technology), and β-actin (bs-0465R; Bioss). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Blots were assessed using BeyoECL Plus (Beyotime). The results were analysed using ImageJ (version 1.4.3.67) software [26 (link)].

The immunohistochemistry in this experiment included the following antibodies: caspase-3 (CASP3; GB11009-1; Servicebio, Wuhan, China; 9 rats from each of the CA and CM groups); interleukin-6 (IL-6; GB11117; Servicebio; 13 and 14 rats from the CA group and CM groups, respectively); mammalian target of rapamycin (mTOR; GB11117; Servicebio; 13 rats from each of the CA and CM groups); nuclear factor-kappa B (NF-κB; 13,533–1- AP; Proteintech, Wuhan, China; 9 rats from each of the CA and CM groups); and tumor necrosis factor-alpha (TNF-α; bsm-33207 m; Bioss, Beijing, China; 13 and 14 rats from the CA group and CM groups, respectively). The experimental method of this part is in the Additional file 3: Experimental methods.