Protein Expression Analysis by Western Blotting

Protein expression was determined by Western blotting, which was performed according to the method of Lee et al. [49 (link)]. Briefly, proteins from cellular lysates were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Danvers, MA, USA). After blocking for 1 h with 5% skim milk in TBST buffer, the membranes were incubated overnight at 4 °C with antibodies specific for the following proteins: iNOS (BD Biosciences, San Jose, CA, USA), COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD80, CD86, TNF-α, phosphor-PI3K p85 (Tyr458), PI3K, phosphor-AKT (T308), AKT, phospho-p70S6K (T389), p70S6K, phosphor-NF-κB (Ser536), NF-κB, PDK1, LDHA (Cell Signaling Technology; Danvers, MA, USA), HIF1-α (Novus Biologicals, Building IV Centennial, CO, USA), and β-actin (Sigma). After three washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies (GeneTex, Irvine, CA, USA). HRP was detected using the ECL reagent (BioNote, Hwaseong, Gyeonggi-do, Korea).