Ab134157

After removing the culture medium, 500 μl protein lysate is added to each well for lysis for 30 minutes, centrifuged at 4° C, 1500 r/min for 5 minutes. And the supernatant is taken to detect the protein concentration AGING of the sample. Transfer the protein to PVDF membrane through Gels preparation, samples loading, electrophoresis and wet transfer and close the blocking solution for 1 hour. Sequentially, primary antibody SHP2 (Abcam, ab187040, 1:5000), Ki-67 (Abcam, ab231172, 1:5000), p-STAT1 (Abcam, ab109461, 1:1000), p-STAT3 (Abcam, ab76315, 1:1000), p-STAT5 (Abcam, ab278764, 1:1000), p-STAT6 (Abcam, ab263947, 1:1000), IL-4 (Abcam, ab34277, 1:1000), IL-10 (Abcam, ab52909, 1:1000), Cathepsin-L (Abcam, ab200738, 1:1000), Cathepsin-S (Abcam, ab134157, 1:1000), Cathepsin-K (Abcam, ab207086, 1:1000), Arginase-1 (Abcam, ab133543, 1:1000) and GAPDH (Abcam, ab181603, 1:10000) are added and the secondary antibody is incubated for 2 hours at room temperature. Exposure imaging is performed and Quantity One V4 software is used for analysis.

Cells were harvested in radio immunoprecipitation assay (RIPA) buffer (Beyotime) containing phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 1 3
(SDS-PAGE). Immunoblotting was performed with primary antibodies including: anti-SNAI1 antibody (1:1000, 3879, Cell Signaling Technology), anti-MMP2 antibody (1:1000, 10373-2-AP, Proteintech), anti-MMP9 antibody (1:1000, 10375-2-AP, Proteintech), anti-FURIN antibody (1:1000, 18413-1-AP, Proteintech), anti-CTSK antibody (1:1000, 11239-1-AP, Proteintech), anti-CTSS antibody (1:1000, ab134157, Abcam), anti-FN antibody (1:1000, 15613-1-AP, Proteintech), anti LAMB1 antibody (1:1000, 23498-1-AP, Proteintech), anti COL IV antibody (1:1000, 55131-1-AP, Proteintech) and anti-β-ACTIN antibody (1:5000, A5316, Sigma-Aldrich) on polyvinylidene fluoride (PVDF) membranes followed by incubation of horseradish peroxidase (HRP)-linked goat anti-rabbit IgG or horse anti-mouse IgG secondary antibody (1:2000, 7074 or 7076, Cell Signaling Technology). Each step was followed by three washes in PBS containing 0.1% Tween-20 (PBST) for 10 min. Membranes were observed using an enhanced chemoluminescence system (Clinx Science Instruments).