Goat anti rabbit igg

MM cells were harvested, washed, and lysed using RIPA lysis buffer (#FMS-WB035, Fcmacs Biotech Co., Ltd., Nanjing, China). Protein concentration were detected by the BCA protein assay kit (#P0011, Beyotime Biotechnology, Shanghai, China). Total protein samples (20–40 μg) were heated in SDS/β-mercaptoethanol buffer and loaded on 10–15% SDS-gels. The proteins were resolved using SDS-PAGE, and then transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk at room temperature for 2 h and incubated with primary antibodies against G6PD (1:1,000 dilution, #ab210702, Abcam, Shanghai, China), β-catenin (1:1 000 dilution, #51,067-2-AP, Proteintech Group, Wuhan, China) and β-actin (1:1000 dilution, #4970S, Cell Signaling Technology, Mass, USA) overnight at 4 °C. Blots were incubated with secondary antibodies using horseradish peroxidase-conjugated rabbit anti-mouse (1:10,000 dilution, #S0002, Affinity, OH, USA) or goat anti-rabbit IgG (1:10,000 dilution, #FMS-Rb01, Fcmacs, Nanjing, China) at room temperature for 1 h. Finally, blots were developed using a chemiluminescence ECL kit (#180-5001, Tanon, Shanghai, China).

Protein levels in MM cells were detected by western blot method, which was performed as previously described (27 (link)). MM cells were harvested, washed and lysed with assistance of RIPA Lysis Buffer. 40 μg total protein samples were heated in SDS/β-mercaptoethanol buffer and loaded on 12–15% SDS-PAGE gels. Proteins were separated by electrophoresis in the gels, and then transferred onto PVDF membrane. The membrane was blocked with 5% non-fat milk and incubated with primary antibodies against Bcl-2 (Transduction laboratones, #610538, 1:1,000 dilution), Bad (Cell Signaling Technology, #9292, 1:1,000 dilution), PARP (Cell Signaling Technology, #9542S, dilution rate1:1000), Caspase 3 (Cell Signaling Technology, #9662s, 1:1,000 dilution), cytochrome C (Cyt C; Cell Signaling Technology, #4272, 1:1,000 dilution), and β-actin(Cell Signaling Technology, #4970S, 1:1,000 dilution) overnight at 4°C. Blots were incubated with secondary antibodies used horseradish peroxidase conjugated rabbit anti-mouse (Affinity, #S0002, 1:10,000 dilution) or goat anti-rabbit IgG (Fcmacs, #FMS-Rb01, 1:10,000 dilution) for 1 h at room temperature. Finally, blots were developed by chemiluminescence using ECL kit (Tanon, Shanghai, China).