Ab9720

The cells were lysed using radioimmunoprecipitation assay lysis buffer (Solarbio Science and Technology Corporation, Beijing, China). The protein concentration was estimated using a bicinchoninic acid protein assay kit. Next, 50 μg of protein was dissolved in 2 × sodium dodecyl sulfate (SDS) loading buffer and boiled at 100°C. After 5 min, the protein was separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk powder at ambient temperature for 1 h and incubated at 4°C overnight with the following primary rabbit antibodies purchased from Abcam: CXCL9 (ab9720, 0.3 μg/ml) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab181602, 1:10,000). The membrane was rinsed with Tris-buffered saline and Tween 20 (TBST) for three times, subsequently incubated with horseradish peroxidase-labeled secondary IgG antibody (1:1,000) for 1 h, and rinsed with TBST three times. The membrane was placed on a clean glass plate, developed with enhanced chemiluminescence kit (BB-3501, Amersham Pharmacia Biotech, Little Chalfont, Bucks, United Kingdom), and photographed by IS gel image analysis system (Alpha Technologies Services Inc., Hudson, OH, United States). The results were analyzed using ImageJ software.