Electrophoresis tank

Extraction buffer comprised of radioimmune precipitation assay buffer, protease inhibitor and phosphatase inhibitor was used on cell pellets with approximately 1 × 10⁶ cells. Protein concentration of the lysate was determined using Pierce BCA protein assay kit. In total, 25 ng of total protein per well was added to 10× loading buffer prior to denaturing at 95 °C for 5 min. Protein was stored at −20 °C prior to use. Gels were hand cast at a concentration of 12–15%. A total of 25 ng of protein was added to each well and electrophoresis was undertaken in a Bio-Rad electrophoresis tank. A current of 15 mAmp per gel was applied for 1 h 20 min. Protein was then transferred to a nitrocellulose membrane with pore size of 0.2 µm prior to Ponceau S staining, Tris buffered saline-Tween (TBST) wash and membrane block in 5% milk and incubation with primary antibody overnight at 4 °C. Further washing with TBST was conducted, then incubation by conjugated antibody for 1 h prior to addition with chemiluminescence substrate and development of photographic film in a dark room. ImageJ software was used to obtain densitometry readings on each blot of interest.

1.5% Agarose (Solarbio) was dissolved in .5×TBE (Solarbio) under a heating condition, and then cooled to form a gel. The products of RT‐qPCR assays and 6× loading buffer were mixed and added into the pores of gel, which was placed in the electrophoresis tank (Bio‐RAD) filled with .5×TBE. Then the electrophoresis tank worked at 130 V for 40 min. The results were obtained via an imaging system (Proteinsimple).

All 2DE chemicals and Ready Strip™ IPG strips were provided by GE Health Life Science. Prior to 2DE, the total protein concentration of the samples was determined by Bradford assay. The 2DE procedure was handled with 3 replications of normal and OCD samples. The 1^st dimension, isoelectric focusing (IEF) was carried out by the application of Bio-Rad Protein IEF Cell, 7cm nonlinear IPG with the pH range of 3 to 11. In this step, about 500μg protein was loaded for each gel. The IEF separates proteins based on their pI. In the 2^nd dimension, proteins were separated based on Molecular Weight (MW) by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and buffering systems in electrophoresis tank (Bio-Rad). After electrophoresis, the gels were dyed by Coomassie blue stating method and, then, scanned using Bio-Rad scanner (Hasanzadeh, Rezaie-Tavirani, Seyyedi, & Emadi, 2015 ). Finally, Progenesis SameSpots software was employed to analyze protein expression changes. A value of 1.5-fold increase or decrease was used as a cut off. Statistically significant differences (P≤0.05) in spot intensities were identified using 1-way ANOVA analysis. The significantly altered proteins in the expression were identified by http://world-2dpage.expasy.org/swiss-2dpage/. The amounts of MW and pI were the indices to identify proteins from this database.

Bradford Protein Quantitative Kit (Shanghai Biyuntian Biotechnology Co., Ltd.) was used to prepare bovine serum albumin (BSA) standard protein solution according to the instructions, and the gradient range was 0–0.5 g/μl. The BSA standard protein solution and the diluted sample were added to the 96‐well plates. Three duplicate wells were made. G250 staining solution of 180 μl was added quickly and placed at room temperature for 5 min. The absorbance at 595 nm was measured. The standard curve was drawn and the protein concentration of the sample was calculated. Twenty micrograms of protein samples were taken for 12% sodium dodecyl‐sulfate polyacrylamide gel electrophoresis by Electrophoresis Instrument (Bio‐Rad Company) and Electrophoresis Tank (Bio‐Rad Company). The concentration of gel electrophoresis was at 80 V and 20 min, and the separation of gel electrophoresis was at 120 V and 90 min. Coomassie brilliant blue R‐250 staining was performed and decolorized until the bands were clear after electrophoresis.