2.6 Western blot assay
The tissue was placed in a precooled homogenizer, and a lysis solution
containing a protease and phosphatase inhibitor was added. After lysis
for 15 min, the sample was centrifuged and stored at -80 °C. The protein
concentration was determined according to BCA. SDS–PAGE electrophoresis
(100 g/L) was carried out after protein denaturation. The proteins were
electrophoresed to a PVDF membrane, which was blocked with BSA reagent
for 1 h. Primary antibodies [anti-MCP1 (bs-1955R, Bioss Antibodies),
anti-MIP2 (bs-1162R, Bioss Antibodies), and anti-KC (AB206411, Abcam),
1:1000] were added and incubated at 4 °C overnight, and the membranes
were washed 5 times with TBST. Then, an HRP-labelled secondary antibody
was added and incubated at room temperature for 1 h. The sample was
developed with a Pierce ECL chemiluminescent kit.