Akt and p akt ser473

For immunoblot analysis, macrophages were lysed in 1× laemmli buffer and the protein concentration was determined using Bicinchoninic Acid Protein Assay (BCA, Sigma-Aldrich). Equal amounts of protein was separated by electrophoresis on 10% SDS-PAGE gel, transferred to PVDF membrane and subsequently probed with the respective antibodies as per manufacturer recommendations. In the present study, the following primary antibodies were purchased either from Sigma Aldrich, Merck [FKHRL1-D12 (p-FOXO3^Thr32), FKHRL-1 (FOXO3), β-actin] or from Cell Signaling Technology, USA (Akt and p-Akt^Ser473). Western blots were quantified by densitometric analysis using Image J software.

For immunoblot analysis, macrophages were lysed in 1× Laemmli buffer and the protein concentration was estimated using Bicinchoninic acid protein assay kit (BCA, Sigma Aldrich, USA). For immunoblot analysis, equal amounts of whole-cell lysate were resolved by electrophoresis on 10% SDS-PAGE gel, transferred to PVDF membrane and subsequently probed with the respective antibodies as per manufacturer recommendations. The primary antibodies used in the study were purchased from either Sigma Aldrich, Merck [FKHRL1-D12 (p-FOXO3Thr32), FKHRL- 1 (FOXO3), β-actin or Cell Signaling Technology, USA (Akt and p-AktSer473).

Total protein concentration of homogenate was measured using a BCA protein assay kit (Beyotime Biotech Inc., Shanghai, China) according to the manufacturer's protocol and was equalized before electrophoresis. Briefly, 40 μg of the proteins in the supernatant was separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking at room temperature for 1 h with 5% nonfat dry milk, the membranes incubated with antibodies TGF-β1 (1 : 500 dilution, Wanleibio, Shijiazhuang, China), PTEN (1 : 400 dilution, Wanleibio, Shijiazhuang, China), PI3K p85 (1 : 1000 dilution, Cell Signaling Technology, USA), p-PI3K p85 (Tyr458)/p55 (Tyr199) (1 : 1000 dilution, Cell Signaling Technology, USA), and Akt and p-Akt (Ser473) (1 : 1000 dilution, Cell Signaling Technology, USA) overnight at 4°C. After washing with TBST, the membranes were incubated with IgG-HRP (1 : 5000 dilution, Wanleibio, Shijiazhuang, China) for 1 h at room temperature. The membranes were developed with enhanced chemiluminescence using ECL reagents (Beyotime Biotech Inc., Shanghai, China) and visualized using a digital imaging system (Bio-Rad Laboratories, Inc., USA). The blots were quantitated by densitometric analysis using NIH ImageJ software. The data were normalized on the basis of GAPDH level.