Quantitative Western Blotting Protocol

Western blotting was performed as previously described^{59 (link)}. Proteins were extracted from cells/tissue using RIPA lysis buffer. The protein concentrations were assayed by bicinchoninic acid method. Equal amounts of protein (20–40 μg) was separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes. Membrane was incubated with a 1:1000 dilution of primary antibody at 4 °C overnight, followed by a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by enhanced chemoluminescence system. Primary antibodies and dilutions used were as follows: SNRK (1:1000; ab96762, Abcam), MCP-1 (1:1000; ab7202, Abcam), TNF-α (1;1000; 3703, Cell Signal Technology), Col I (1:1000; 84336, Cell Signal Technology), and Col IV (1:200; ab6586, Abcam). GAPDH was used as an internal reference. The image collection and quantitative analysis were performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA USA). Uncropped immunoblot gels are displayed in Supplementary Fig. 19.