Autoradiography film

For immunoblot analysis, cells were lysed in LDS sample buffer (Invitrogen) at each time point. Activation of Dectin-1 signaling pathways was measured by immunoblotting with antibodies against phospho-SYK (Y525/Y526), SYK, pERK (Thr202/Tyr204), ERK (all from Cell Signaling), and GAPDH (Santa Cruz). Immunoblots were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and by exposure to autoradiography film (VWR) or with ChmiDoc (Bio-rad). ImageJ was used for quantification of the band intensity.

An equal volume of protein was size-separated by electrophoresis on SDS-polyacrylamide gels and electroblotted onto polyvinylidene fluoride transfer membranes (PVDF) (Bio-Rad Laboratories, Hercules, CA, USA). After 1 h of blocking at room temperature with 5% skim milk in PBS 0.1% Tween, blots were incubated with the specific primary antibodies overnight at 4°C. Then, membranes were treated with the appropriate secondary antibody for 1 h at room temperature. All blots were treated with Immobilon ECL Western Blotting Detection Kit Reagent (EMD Millipore, Billerica, MA, USA) and developed after exposure to an autoradiography film (VWR International, Radnor, PA, USA). The primary antibodies used were Phospho-Akt (#9271), Akt (#9272), Phospho-mTOR (#5536) and procaspase 3 (#9662) from Cell Signaling (Beverly, MA, USA); FOXO3a (#06-951) from Upstate (EMD Millipore); Phospho-FOXO3a (sc-101683), Phospho-JNK (sc-6254), FOXM1 (sc-500), Bax (sc-493), CDK4 (sc-260), CDK6 (sc-177), ERK 2 (sc-154) and Lamin B (sc-6217) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Phospho-PRAS40 (#44-1100) from BioSource International (Camarillo, CA, USA); PARP (#556493) and cytochrome c (#556433) from BD Pharmingen (BD Biosciences); p27 (#610242) from BD Transduction Laboratories (BD Biosciences) and β-actin (#69100) form MP Biomedicals (Santa Ana, CA, USA).