Ecl buffer

Myocardial tissue and cell suspension was lysed by RIPA lysis buffer (MB-030-0050, Multi Sciences Biotech, Hangzhou, China). BCA Protein Assay Kit (23225, Thermo Fisher, USA) was adopted to determine protein concentrations. After equal quantities, total proteins were separated in SDS-PAGE gel (4-20%, ACE Biotechnology, Xiangtan, China), and the bands were transferred onto PVDF membranes (03010040001, Merck, USA). After blocked with 5% skimmed milk and washed with tris-buffered saline containing 0.1% tween-20 (TBST), protein bands were blotted with primary antibodies at 4° C overnight. After washing with TBST, protein bands were blotted with HRP conjugated secondary antibody and monitored using ECL buffer (32209, Thermo Fisher). Quantifications of western blotting were measured with Fusioncapt advance software. Antibody information: Bax (HUABIO, USA, ET1603-34, 1:1000), Bcl2 (HUABIO, ET1702-53, 1:1000), Cleaved-caspase3 (HUABIO, ET1608-64, 1:1000), IL-10 (Servicebio, Wuhan, China, GB11108, 1:1000), TLR4 (Servicebio, GB11519, 1:1000), α-SMA (Servicebio, GB111364, 1:1000), MMP-9 (Abcam, Cambridge, UK, ab76003, 1:1000), Collagen I (Abcam, ab34710, 1:1000), GAPDH (CST, USA, 5174S, 1:1000). HRP-conjugated secondary antibody (HUABIO, HA1001, and HUABIO, HA1006, 1:50000).

Myocardial tissue and cell suspension were lysed by RIPA lysis buffer (MB-030–0050, Multi Sciences Biotech). BCA Protein Assay Kit (23225, ThermoFisher) was adopted to determine protein concentrations. After equal quantities, total proteins were separated in SDS-PAGE gel (4–20%, ACE Biotechnology), and the bands were transferred onto PVDF membranes (03010040001, Merck). After being blocked with 5% skimmed milk and washed with tris-buffered saline containing 0.1% tween-20 (TBST), protein bands were blotted with primary antibodies at 4 °C overnight (antibody information is provided in Supplementary Table 3). After washing with TBST, protein bands were blotted with HRP-conjugated secondary antibody and monitored using ECL buffer (32209, ThermoFisher). Quantifications of Western blotting were measured with Fusioncapt advance software.

Skin tissues and cell suspension were lysed by RIPA lysis buffer (MB–030–0050, Multi Sciences Biotech, Hangzhou, China) complemented with phenylmethylsulfonyl fluoride. A BCA Protein Assay Kit (23225, Thermo Fisher, Waltham, MA, USA) was adopted to determine protein concentrations. After establishing equal quantities, total proteins were separated in SDS–PAGE gel (4–20%, ACE Biotechnology, Nanjing, China), and the bands were transferred onto PVDF membranes (Millipore, NJ, USA). After blocking with 5% skimmed milk and washed with tris–buffered saline containing 0.1% tween–20 (TBST), protein bands were blotted with primary antibodies at 4 °C overnight (Antibody information is provided in Supplementary Table S2). After washing with TBST, protein bands were blotted with HRP conjugated secondary antibody and monitored using ECL buffer (32209, ThermoFisher, Waltham, MA, USA). Quantifications of western blotting were measured with ImageJ.

RIPA Lysis Buffer was used to extract cell proteins. Gel electrophoresis was performed, and proteins were transferred to the PVDF membrane (Millipore USA). Then, the membrane was blocked for 2 h at room temperature and incubated with 1 ∶ 1000 GAPDH, iNOS, IL-6, RhoA, ROCK1, ROCK2, NF-κB, IκB, and phosphorylated-IκB antibodies at 4°C overnight. Subsequently, HRP-conjugated Goat anti-rabbit antibody (1 : 10000) was added and incubated for 2 h at room temperature. The membrane was detected with ECL buffer (Thermo Fisher Scientific, USA) by the Chemiluminescence imager (Tanon, China). Protein levels were normalized to GAPDH. The expression of phospho-IκB was measured by the ratio of phospho-IκB to total IκB.