Anti ccrl2

Western blotting was performed on cytosolic cellular extracts. Cells were washed with cold phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of lysis buffer containing protease and phosphatase inhibitors. Cell lysates were clarified by centrifugation (4 °C, 15 min, 12,000 rpm), and protein was subjected to 10% sodium dodecyl sulfate-PAGE (SDS-PAGE) and transferred to a nitrocellulose membrane using a wet transfer system. Membranes were incubated with 5% skim milk dissolved in TBS plus 0.05% Tween 20 (TBST) for 1 h to block nonspecific protein-binding sites. Then, the membranes were incubated with anti-CCRL2 (Abcam, no. ab88632), anti-CX3CR1 (Abcam, no. ab8021), anti-p38 MAPK (Abcam, no. ab197348), anti-ERK (Abcam, no. 196883), anti-AKT (Abcam, no. ab8805), anti-phospho-ERK (Abcam, no. ab50011), anti-phospho-AKT (Abcam, no. ab81283), anti-phospho-p38-MAPK (Abcam, no. ab47363), and anti-GAPDH (Abcam, no. ab181602) antibodies at 4 °C overnight according to the manufacturer’s instructions. The membranes were then washed with TBST and incubated with a secondary anti-rabbit Ab conjugated to HRP (Cell Signaling Technology, no. 7074s) at room temperature. The signals were detected and analyzed by a chemiluminescence imaging system (ChemiScope5600, CLINX, Shanghai, China), and each experiment was performed in triplicate.