Versadoc 5000 system

Western blots were performed using 3T3-L1 cells. Whole cell extracts were prepared in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.5% sodium deoxycholate, and 0.1% SDS). Protein concentration was determined by the Bradford method using β-globulin as a standard. Twenty microgram of protein was prepared in sample loading buffer containing β-mercaptoethanol heated at 95°C for 10 min, then resolved on 12% SDS-PAGE gels. Proteins were then transferred at 100 V for 75 min onto PVDF membranes, blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 h at RT, and then probed with the anti-UCN3 primary antibody (1:1,000 dilution) (bs-2786R, Bioss Antibodies Inc., MA, USA) for overnight at 4°C. After washing, the membranes were incubated with Rabbit horseradish peroxidase conjugated secondary antibody (1:10,000 dilution) for 2 h at RT, and finally detection was performed using super sensitivity West Femto ECL reagent (Thermo Scientific, USA). Protein bands were visualized by chemiluminescence and the images captured by using the Versadoc 5000 system (Bio-Rad, USA). GAPDH was used as internal control to monitor for protein loading, with the anti-GAPDH (ab2302, Millipore, Temecula, CA). For densitometric analysis, the intensity of bands was determined using Quantity One Software (Bio-Rad, USA).

Western blot analysis was performed using whole PBMC extracts prepared in RIPA buffer (50 mM Tris HCL pH 7.5, 150 mM NaCl, 1% Triton × 100, 1 mM EDTA, 0.5% Sodium deoxycholate, and 0.1% SDS). Protein concentration was determined using the Bradford method with bovine serum albumin (sigma Aldrich) dissolved in RIPA as a standard. The protein samples (20 μg) were resolved in 10% acrylamide gels, then transferred onto PVDF membranes, and blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.05% Tween 20 for 2 h at room temperature. The membranes were incubated with primary antibody against DNAJC27 diluted 1:1000 (Abgent Cat. # AP12755a, San Diego, California, USA) overnight at 4°C. The next day, the membranes were washed and incubated with secondary anti-rabbit antibody (GE Amersham Cat. # NA9340) with a 1:10,000 dilution). Protein bands were visualized using chemiluminescence (Super signal, Thermo Fischer) and images were captured using Versadoc 5000 system (Bio-rad, Hercules, CA).

Target proteins were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The cells were lysed using cell lysis buffer (Sigma-C2978) with protease inhibitors (Complete Protease Inhibitor Tablets; Boehringer Mannheim, Indianapolis, IN, USA). The proteins were separated using 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight at 4 °C with a primary antibody against a specific target and chemiluminescent detection was uses a horseradish peroxidase-conjugated secondary antibody (1:5000). The signals were visualized with a chemiluminescence reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and detected using the VersaDoc 5000 system (Bio-Rad Laboratories, Hercules, CA, USA) [33 (link)].