Biorad chemidox

HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) for 48 hours. Cells were collected using RIPA lysis buffer (R&D) and proteins were obtained by centrifugation. Proteins were incubated with protein A + G agarose beads (Beyotime) and antibodies overnight. Immunoprecipitated proteins were collected after washing three times with PBS (Sigma). The bicinchoninic acid assay (Pierce) was used to measure protein concentration. Proteins were denatured at 100 °C for 5 minutes and separated in 10% SDS-PAGE at 80 mA for 2 hours. They were then transferred to PVDF membrane (Millipore) at 100 mA for 2 hours. The membrane was blocked in 5% BSA (Sigma) in TBS (Sigma) with 0.1% Tween 20 (TBST) at room temperature for 1 hour. The antibodies were diluted at suitable concentration and added into TBST with 5% BSA. Primary antibodies were incubated overnight at 4 °C. HRP-conjugated second antibodies were added after washing three times for 10 minutes by TBST. Enhanced chemiluminescence was used to detect optical density of HRP after washing three times for 10 minutes. BioRad ChemiDox (BioRad) was used to analyze optical density of HRP.

Tissue samples were collected using RIPA lysis buffer (R&D), and proteins were obtained by centrifugation. Proteins were denatured at 100°C for 5 min and separated in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) at 80 mA for 2 h. They were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) at 100 mA for 2 h. The membranes were blocked in 5% bovine serum albumin (BSA; Sigma) in Tris‐buffered saline (TBS; Sigma) with 0.1% Tween 20 (TBST) at room temperature for 1 h. Antibodies were diluted at a suitable concentration and added to TBST with 5% BSA. Primary antibodies were incubated overnight at 4°C. Horseradish peroxidase (HRP)‐conjugated second antibodies were added after washing three times for 10 min each time with TBST. Enhanced chemiluminescence was used to detect the optical density of HRP after washing three times for 10 min each time. BioRad ChemiDox (BioRad) was used to analyze the optical density of HRP.

Proteins from the cultured cells were obtained using cell lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% (w/v) NP-40, and 0.1% (w/v) SDS). Protein concentrations were measured with a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein extracts were denatured at 100 °C for 5 min and separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for ~ 1.5 h. Proteins were blotted onto an Immobilon-P™ polyvinylidene fluoride (PVDF) membrane (Merck Millipore Ltd., Dublin, Ireland) at 100 V for 2 h. The membranes were blocked with 5% (v/v) bovine serum albumin (BSA) in Tris-buffered saline with 0.1% (w/v) Tween-20 (TBST) at room temperature for 30 min. The membranes were then incubated overnight at 4 °C with primary antibodies to various candidate proteins (1:1,000). The membranes were washed thrice with TBST. Specific horseradish peroxidase (HRP)-conjugated secondary antibody was added at 1:5000 and detected by enhanced chemiluminescence (ECL). The optical density was then measured by BioRad ChemiDox (BioRad Laboratories, Hercules, CA, USA).