Enhanced chemiluminescence detection system on radiograph film

Total protein was extracted from freshly isolated or cultured B cells and the concentration was determined with the BCA protein concentration assay kit. Sample protein was separated by electrophoresis in 10% SDS-PAGE with the Bio-Rad electrophoresis system (Hercules, CA, USA). The primary antibodies (rabbit anti-PTEN, PI3K p85α, total-AKT1/2/3, p-AKT (ser473), TLR-2, TLR-4, Abcam, UK, 1:1000 dilutions) were incubated at 4 °C overnight. The secondary antibodies (anti-rabbit IgG, 1:2000 dilutions) were incubated for 2 h at room temperature. The membrane containing antibody-protein complexes were visualized with an enhanced chemiluminescence detection system on radiograph film (Bio-rad, Hercules, CA, USA). The bands were scanned and analyzed by the software Quantity ONE (Bio-rad, Hercules, CA, USA). The expression of protein in each sample was normalized by GAPDH.

Total protein was extracted from MIBECs and analyzed with bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Sample protein was separated by electrophoresis in 12 % SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). The primary antibodies (rabbit anti-TLR4, at 1 μg/ml, NF-κB p-65 at 1 μg/ml, MyD88 at 1 μg/ml antibody, Cell Signaling biotech Co, Ltd, MA, USA, 1:1000 dilutions) were incubated at 4 °C overnight. The secondary anti-bodies (anti-rabbit IgG, 1:5000 dilutions) was incubated for 1 h at room temperature. The membrane containing antibody-protein complexes were visualized with an enhanced chemiluminescence detection system on radiograph film (Bio-rad, Hercules, CA, USA). The bands were scanned and analyzed by the software Quantity ONE (Bio-rad, Hercules, CA, USA). The expression of protein in each sample was normalized by α-Tublin (Santa Cruz Biotechnology, CA, USA).