Cxcl1

The protein lysates were prepared using RIPA buffer, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and probed with primary antibodies at 4 °C overnight. The primary antibodies including vimentin (no. A2666), N-cadherin (no. A0443), Arg-1 (no. A1847), CXCL1 (no. A5802), SOX4 (no. ab80261), p-P65 (no. AP0475), P65 (no. A2547), IKKα (no. A2062), IKKβ (no. A2087) were provided by ABclonal Technology (Cambridge, MA, USA). E-caherin (no. 3195s), p-IKBα (no. 2859), IKBα (no. 4814), p-IKKα/β (no. 2697), β-actin (no. 4970S) and GAPDH (no. 5174s) were purchased form Cell Signaling Technology (Beverly, MA, USA). β-Catenin (sc-7199) and iNOS (bs-0162R, BIOSS) were bought from Santa Cruz (Santa Cruz, CA, USA) and BIOSS (Woburn, MA, USA), respectively. CXCR2 (ab14935) was provided by Abcam (Cambridge, MA, USA). After three washes with Tris-buffered saline with 0.05% Tween-20, the membranes were incubated with secondary anti-rabbit or anti-mouse antibodies (Cell Signaling, MA, USA) for 1 h at room temperature. The signals were visualized using the ECL Advance reagent and quantified using ImageLab software. A triplicate independent experiment was performed.

Proteins were extracted from heart tissue or cells using RIPA lysis buffer (Millipore, HY-K0010) with a proteinase inhibitor cocktail (Millipore, HY-K0010). The protein was separated by 10% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore, IPFL00005, United States). Membranes were blocked with 5% non-fat milk in TBST at room temperature for 60 min and incubated overnight with indicated primary antibodies at 4°C, followed incubation with HRP-linked secondary antibodies at room temperature for 60 min, and the specific proteins were detected by an ECL Detection System (Pierce, Rockford, IL, United States). The primary antibodies used in Western Blot include KLF15 (Proteintech, 66185, China), P-mTOR (CST, 5536, United States), mTOR (CST, 2983, United States), P-NF-κB p65 (CST, 3039, United States), NF-κB p65 (CST, 8242, United States), P-ERK1/2 (CST, 4370, United States), ERK1/2 (CST, 4695, United States), CXCL1 (ABclonal, A5802, China),CXCR2 (ABclonal, A3301, China), GAPDH (CST, 8884, United States), Flag (Sigma, F1804, United States), and anti-rabbit or anti-mouse secondary antibodies (CST, 7074 or 7076, United States).