Rabbit anti s100a4

RIPA lysis buffer that contained a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai) was utilized to lyse cells and lung tissues for thirty minutes on ice. The samples were centrifuged at a centrifugation rate of 12,000 g for thirty minutes to sediment the debris. A BCA Assay Kit (Solarbio Life Science, Beijing, China) was used in order to assess the protein content. Proteins (30μg) were isolated using 10% SDS-PAGE and put into PVDF membranes. These plots were then subjected to incubation overnight at a temperature of 4°C with rabbit anti-α-SMA (1 : 500; Abcam), rabbit anti-S100A4 (1 : 1000; Abcam), rabbit anti-p-ERK1/2 (1 : 1000; Cell Signaling Technology), rabbit anti-fibronectin (1 : 1000; Abcam), rabbit anti-ERK1/2 (1 : 1000; Cell Signaling Technology), rabbit anti-FAK (1 : 1000; Cell Signaling Technology), rabbit anti-p-FAK (1 : 1000; Cell Signaling Technology), rabbit anti-p-GSK-3β (1 : 1000; Cell Signaling Technology), and rabbit anti-GSK-3β (1 : 1000; Cell Signaling Technology). As an internal control, GAPDH (1 : 10000; Abcam) was employed. After the membranes were washed, they were exposed to incubation for one hour at room temperature with HRP-conjugated secondary antibodies. The ImageJ program (version: 1.46) from the National Institutes of Health, Bethesda, MD, was utilized for the purpose of conducting the densitometric analysis.

Immunostaining was performed as described previously [53] (link) using the following primary antibodies: rat anti-CD3 (clone 17A2, BD Biosciences, 1∶500), rat anti-Ly6G (clone 1A8, BD Biosciences, 1∶5000) and rabbit anti-S100A4 (Abcam, 1∶200).