Bandscan software

Total protein was extracted by lysing cells in RIPA buffer containing protease inhibitor. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidenefluoride (PVDF) membranes. After blocking with 5 % non-fat milk or 3 % BSA in TBS-T, membranes were incubated with the primary antibody. The following antibodies were used: SH2B1 (1:1000,Abcam, USA), JAK2 (1:600, Abcam, USA), p-JAK2 (1:2000, Abcam, USA), p-Rac1 (1:500, Abcam, USA), Anti-cAMP Protein Kinase Catalytic subunit (1:60000,Abcam,USA),MMP2 (1:2000,Abcam.,USA), MMP9 (1:1000, Abcam,USA), GAPDH (1:10000, Abcam, USA) and goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1:5000, Santa Cruz, USA),which was used as the secondary antibody. Cells were seeded on 10 cm cell culture plates, grown to 80 % confluences, and serum starved overnight. Target signals were quantified by BandScan software (Bio-Rad, Hercules, CA) and defined as the ratio of target protein relative to GAPDH.

Murine blood samples were homogenized in RIPA Buffer (R0010, Solarbio, Beijing, China) according to the manufacturers’ protocols. Protein concentration of the lysates was determined by BCA Protein Assay Kit (PC0020, Solarbio, China). Equal numbers of protein samples (15 μg/lane) were separated using 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (88585, Thermo Fisher, USA), followed by treatment with blocking buffer (P0252, Beyotime, Shanghai, China). Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight. The next day, hybridization with horseradish peroxidase-conjugated secondary antibodies (A0192, A0208, Beyotime, China) was performed at room temperature (RT) for 1 h. The stripped membranes were subsequently treated with ECL Western Blotting Detection Kit (SW2040, Solarbio, China), and immunoblots were detected by ChemiDoc XRS + imaging system (BIO-RAD, Hercules, CA, USA). Relative protein expression represented as the ratio of the gray-scale value of the target band to that of the GAPDH band was analyzed by Bandscan software (BIO-RAD, USA).

Total protein was separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, MA). The blocked membranes were incubated with the primary antibodies followed by HRP-conjugated secondary antibodies (KPL, Gaithersburg, MD). Bands were visualized using an enhanced chemiluminescence kit (Santa Cruz Biotechnology, Santa Cruz, CA). The target signals were quantified by BandScan software (Bio-Rad, Hercules, CA) and defined as the ratio of target protein signal relative to β-actin signal. The antibodies used in the study are listed in Additional file 2: Table S3.