Pierce 1 step transfer buffer

The total protein from each group was extracted by RIPA lysate, and the concentration of each group was determined by BCA method (Beyotime, China). The SDS-PAGE gel electrophoresis was performed with 30 μg of protein in each group. After electrophoresis, the target protein gel was cut off from the corresponding position referring to the prestained protein. The target protein was transferred to the PVDF membrane by semidry transfer method (Pierce™ 1-Step Transfer Buffer, Thermo, USA) and blocked at room temperature for 1∼2 h and then hybridized with rabbit anti-rat β-actin antibody (1 : 1000, CST, USA), rabbit anti-rat Fyn antibody (1 : 300, Santa Cruz, USA), and rabbit anti-rat STAT5 antibody (1 : 300, Santa Cruz, USA), and then incubated with goat anti-rabbit HRP-IgG antibody after washing for three times (1 : 5000, Bioss, China). The PVDF membranes were fully washed with TBST, and the images were developed by ECL chemiluminescence (ChemiDoc XRS + Imaging System, Bio-Rad, USA). The gray values of each band were read by Image Lab 4.0 software, and β-actin was used as an internal control to analyze the expression levels.

7.5% polyacrylamide gels were cast and loaded with 50 µg of sample mixed with Laemmli buffer (1:1) that was previously boiled for 5 minutes. Gels were run for 90 minutes at 120 V. Gels were then stained with GelCode (Thermo-Fisher) following manufacturer’s directions. For western blot, 100 µg were loaded into precast 12% SDS-PAGE gels (Biorad) and run at 200 V for 45 minutes. Gels were transferred to a PVDF membrane using a semi-dry transfer system (Thermo-Fisher) with Pierce 1-step transfer buffer (Thermo-Fisher). The membrane was activated with methanol and then blocked overnight at 4 °C with 3% BSA. Primary antibody (keratocan, sc-66941, Santa Cruz) was used at a dilution of 1:200 in 3% BSA and incubated overnight at 4 °C. Three 5-minute washes with TBST were performed under agitation, and then the HRP-linked secondary antibody (A0545, Sigma-Aldrich) at a dilution of 1:1000 was incubated for an hour at room temperature. Another set of washing steps was carried out and then membranes were developed with Western Chemiluminescent HRP Substrate (Fisher Scientific). Membranes were imaged with GelDoc (Biorad).