Ab32828

Axl tyrosine kinase inhibitors R428 (BGB324) and SGI-7079 were purchased from Selleck Inc. R428 and SGI-7079 specificities for Axl have been previously evaluated [21 (link), 24 (link)]. They were dissolved in DMSO at the concentration of 1 mM for in vitro studies while formulated in 0.5% hydroxypropylmethylcellulose plus 0.1% Tween 80 for in vivo studies. Transfection agent Lipofectamine 2000 was obtained from Invitrogen. Scramble control Axl-targeting siRNA was synthesized by Shanghai GenePharma Inc with the following sequences: siAxl-1: 5′-GCCAGATGAAATCCTCTAT-3′, siAxl-2: 5′-CGAGGTACTTATGGATATA-3′; siNC: 5′-AATTGTACTACACAAAAGTAC-3′. Anti-mouse PD-1 (RMP1–14), CD4 (GK1.5), CD8 (2.43), NK 1.1 (PK136) and their isotype control (2A3) antibodies were purchased from BioXCell. Primary rabbit anti-mouse/human Axl antibody (ab32828) and secondary FITC-labelled goat anti-rabbit antibody (ab6717) were obtained from Abcam. PE-conjugated anti-mouse Axl (FAB8541P), Mer (FAB5912P) and rat IgG2a isotype control (IC006P) antibodies were purchased from R&D Systems. The fluorescence labeled monoclonal antibodies (mAbs) for FACS analysis were all from BD Bioscience.

Cells were lysed in RIPA buffer. Twenty micrograms proteins were loaded and then separated on 10% SDS/PAGE gel. PVDF membranes (Millipore, Billerica, MA) were then used for western transfer. After being blocked in 5% milk, the membranes were incubated with appropriate dilutions of specific primary antibodies against GAPDH (Santa Cruz, #sc-166574, Paso Robles, CA), β-actin (Abcam, #ab8227, San Diego, CA), TR4 (Abcam, #ab109301), or AXL (Abcam, #ab32828). After being incubated with HRP conjugated secondary antibodies, the blots were visualized using the ECL system (Thermo Fisher Scientific, Rochester, NY).