Ab109245

Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes (Amersham). Membranes were blocked overnight in 5% w/v BSA and incubated with primary antibody for 2 h. Anti-GST-HRP (RPN1236, 1:10000) was from Sigma-Aldrich. Anti-phosphotyrosine antibody (4G10) was obtained from Millipore. Anti-Elongin C antibody (610761, 1:3000) was from BD Biosciences. Anti-p44/42 (9102S, 1:2000), anti-STAT5 (94205; 1:3000), and anti-phospho-STAT5 (9359; 1:2000) were purchased from Cell Signaling. Anti-actin-HRP antibody (C4) was obtained from Santa Cruz (sc-47778 HRP; 1:1000). Anti-Cullin 5 (ab184177), anti-Elongin B antibody (ab154854, 1:2000), and anti-SOCS2 antibody (ab109245, 1:1000) were purchased from Abcam. Antibody binding was visualized with peroxidase-conjugated sheep antirabbit immunoglobulin (Southern Biotech; 4010-05; 1:15000), or sheep antimouse immunoglobulin (GE Healthcare; NA931-1ML; 1:10000) and the enhanced chemiluminescence (ECL) system (Amersham or Millipore).

This study was approved by the Institutional Review Board of Zhabei Central Hospital of Jing'an District. Paraffin-embedded CRC specimens from patients treated at Zhabei Central Hospital of Jing'an District were enrolled in this study with their written consent. None of these patients had received radiotherapy or chemotherapy prior to surgery.
CRC specimen sections were deparaffinized and dehydrated using sequential diluted xylene and ethanol wash. The antigen was retrieved by treating slides in 0.01 M sodium citrate buffer (pH 6) for 15–20 min. The slides were then permeabilized in 3% hydrogen peroxide and blocked using 5% bovine serum albumin. Primary antibody anti-TRIM6 (ProteinTech, 11953-1-AP), anti-SOCS2 (Abcam, ab109245), or anti-p-STAT3 (Abcam, ab76315) was incubated overnight. The corresponding HRP secondary antibodies (Long Island Bio, Shanghai, China) were then used. The signal was developed using the DAB staining kit (Long Island Bio) along with hematoxylin staining. The intensity grading was calculated as follows: IRS = staining intensity (SI) × percentage of positive cells. The SI was ranked from 1 to 3 (negative to strong). Overexpression was defined as having a staining index >3. Staining was independently assessed by two investigators.

Total proteins were extracted by RIPA buffer, and BCA protein assay kit was used to determine the corresponding concentration. Protein (25 μg) was isolated by 8% SDS–PAGE and subsequently transferred to PVDF membranes. The 1% BSA in TBS buffer was used to block the membranes, and primary antibodies were cultivated at 4°C overnight. The membrane was then washed with 1× TBST and cultivated with a secondary antibody horseradish-peroxidase-conjugated in 1×TBS at room temperature for 1 h. The membrane was washed with 1× TBST, and the protein expression was determined using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). The loading control was β-actin, detected on the same blot. All primary antibodies were purchased from Abcam: DNMT3A (ab188470; 1:2,000), DNMT3B (ab2851; 1:2,000), SOCS2 (ab109245; 1:5,000), STAT5 (ab126832; 1:1,000), P-STAT5 (ab32364; 1:1,000), and the secondary antibody (ab6802, 1:2,000).