Anti hck

Anti Cd11a inhibitory antibodies MHM 24, TS1.22.1.1.13.3 and Tranferrin receptor antibodies G1/221/12 were obtained from the mouse hybridoma bank (DSHB) as cell culture supernatants. Other antibodies and their sources were: anti-M2 FLAG antibody (Sigma), anti-phospho LCK Y394 (Thermo scientific), anti-LYN (Cell Signaling Technologies), anti-HCK (Cell Signaling Technologies), anti-FGR (Cell Signaling Technologies), anti-phospho Src family Y416 (recognizing phosphor-LYN, HCK, and FGR; Cell Signaling Technologies), anti-M2 Flag Beads (Sigma), Glutathione Sepharose (GE healthcare).

For cell sample preparation, cells were lysed with an NP-40 buffer with protease inhibitor cocktail (Roche, 11697498001). For tissue sample preparation, single-cell suspensions derived from mouse tumors were incubated with anti-CD11b antibody–conjugated microbeads (1:100; Miltenyi Biotech, 130-049-601) for 15 min at 4°C and separated by MACS column with a separator. The eluted cells were lysed with NP-40 buffer with protease inhibitor cocktail and phosphatase inhibitor (Thermo Fisher Scientific, 78428). Total protein (20 μg) was resolved by 4 to 15% precast SDS–polyacrylamide gel electrophoresis (Bio-Rad) and followed by transfer. Polyvinylidene difluoride membranes were blotted with anti–phosphoSrc (Tyr⁴¹⁶; Cell Signaling Technology, 6943), anti-Hck (1:1000; Cell Signaling Technology, 14643; ABclonal, A14537), anti-Lyn (1:1000; Cell Signaling Technology, 2796), anti-FLAG (1:1000; GenScript, A00187-100), anti–arginase 1 (1:500; Santa Cruz Biotechnology, sc-20150), or anti–glyceraldehyde-3-phosphate dehydrogenase (1:3000; Cell Signaling Technology, 5174) antibody overnight at 4°C. Proteins were detected with horseradish peroxidase–conjugated secondary antibodies (Bio-Rad) and enhanced by enhanced chemiluminescence development (GE Healthcare, RPN2232).