Anti cx3cr1

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.

Anti-TUBB and anti-ACTB were purchased from Developmental Studies Hybridoma Bank (Iowa City, IA). Anti-rabbit IgG HRP-linked antibody, anti-phospho-CHEK2, anti-phospho-CHEK1, and anti-CHEK1, antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti- CX₃CR1, anti-phospho-ATM, anti-phospho-PRKDC, anti-PRKDC, anti-ATM, and anti-phospho-H2AX antibodies were obtained from Abcam (Cambridge, MA). Anti-mouse IgG HRP-linked antibody, anti-phospho-ATM, anti-CHEK2, anti-MYC, and anti-phospho-H2AX were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG were from ThermoFisher Scientific (Waltham, MA). Anti-GPR65 and anti-FFAR4 antibodies were obtained from One World Lab (San Diego, CA). Anti-RAD50 antibodies were purchased from NeoBiolab (Cambridge, MA).

Selected antigens were labeled with the use of antibodies: anti-CD34 (unconjugated, Dako, Glostrup, Denmark), anti-CX3CR1 (unconjugated, Abcam Inc., Cambridge, MA, USA), anti-cytokeratin 7 (unconjugated, Dako). To visualize presence of unconjugated antibodies Alexa 488 and Alexa 647 were used. DAPI has been used to counterstain nuclei. The staining protocols were based on producer's manuals. We have selected pairs of antigens to be visualized on a single section: CD34 and CX3CR1, and cytokeratin 7 (CK7) and CX3CR1. The slides were investigated and microphotographed on a confocal microscope (FV1000 Olympus).