Biotinylated anti f4 80

Briefly, FVB/N mice received subcutaneous injections of 5 × 10⁶ NT2.5 cells into the right upper mammary fat pad. One week post-tumor challenge, mice were vaccinated with 3 × 10⁶ irradiated 3T3neuGM cells or 3T3GM cells. Sorafenib (30 mg/kg) or vehicle was given by daily gavage starting on the day of vaccination. Tumors were harvested Day 12 post-treatment and suspended in 5 ml RPMI 1640 containing Liberase TM (Roche). Tumors were minced and incubated at 37 °C for 30 min. Suspensions were then passed through a 100-μm mesh nylon cell strainer (BD Falcon) to obtain single cell suspensions. Single cell suspensions were incubated with in ACK lysing buffer (Sigma) for 2 min at room temperature and washed twice in FACS-staining buffer (PBS, 2% heat-inactivated FCS). Pellets were resuspended in FACs staining buffer containing Fc Block (MACs Miltenyi Biotec) and incubated on at 4 °C for 20 min. CD11b⁺ TAMs were obtained through positive isolation (MACs Miltenyi Biotech). CD11b⁺ TAMs were incubated with FACs buffer containing biotinylated anti-F480 (eBioscience) and isolated by positive selection (MACs Miltenyi Biotec).

After 3hr incubation on plate, or isolation of F4/80⁺ cells using biotinylated anti-F4/80 (ebioscience) antibody with MojoSort™ Streptavidin Nanobeads (Biolegend), or after sorting, cells were washed twice with PBS, then peritoneal exudate cells (3 × 10⁶) were lysed in RIPA buffer with NaF, protease inhibitor cocktail (Roche), PMSF, sodium pyrophosphate, beta glycerophosphate and sodium orthovanadate (Sigma Aldrich). Immunoblot was performed using a standard protocol (Life Technologies). The following antibodies were used from Cell Signaling: anti-p-AKT (S473, D9E or D7F10), anti-AKT1 (C73H10), anti-p-S6(Ser240/244, D68F8), anti-S6(5G10), anti-p-4E-BP1(T37/46, 236B4), anti-4E-BP1(53H11), anti-p-FOXO1(Thr24)/FOXO3a(Thr32), FOXO1(C29H4) and anti-β-actin (D6A8), Sigma-Aldrich: anti-actin. All images were captured and analyzed using UVP BioSpectrum 500 Imaging System.