Anti stat3 antibody

Insulin and glucagon contents in pancreatic islets were determined through an immunohistofluorescence analysis. Briefly, fresh pancreatic tissues were fixed in 10% paraformaldehyde and embedded prior to sectioning. All sections were incubated with secondary antibody (Jackson ImmunoResearch, USA) in the dark for 1 hr at room temperature and dyed with DAPI after staining with primary antibody (anti-insulin antibody (CST, USA) and anti-glucagon antibody (Abcam, USA)) at 4°C overnight.
The insulin content was measured, and Stat3 translocation in INS-1 cells was detected through an immunocytofluorescence analysis. INS-1 cells were seeded on circular slides in a 6-well plate. After the corresponding treatment, the cells were fixed in 4% paraformaldehyde for 15 min and blocked with 5% BSA for 30 min, followed by incubation with the corresponding primary antibody (anti-insulin antibody (CST, USA) and anti-Stat3 antibody (Abcam, USA)) at 4°C overnight, and the cells were then incubated with FITC-tagged secondary antibody (Jackson ImmunoResearch, USA) in the dark for 1 hr at room temperature and dyed with DAPI to capture the distribution of fluorescence under a fluorescence microscope (Olympus, Japan).

Curcuma (TCM Pharmacy of Longhua Hospital of Shanghai University of Traditional Chinese Medicine), DSS (MP Biomedicals, USA), absolute ethyl alcohol, Tween-20, xylene substitute (Sinopharm Group Chemical Reagent Co. Ltd.), RIPA Lysis buffer, PMSF, BSA, BCA Protein Quantitation Kit (Beyotime), PAGE gel rapid preparation kit, Multicolor Restrained Protein Ladder (Shanghai EpiZyme Biotechnology Co., Ltd.), β-actin, anti-STAT3 antibody, anti-TNF-α antibody (Abcam Company, England), HE dyeing (Shanghai Yixin Biotechnology Co., Ltd.), and neutral gum (Shanghai Yiyang Instrument Co., Ltd.) were used.

Cells were cultured in a serum‐free medium overnight, then treated with serum‐free medium containing 10 × 10⁻⁹m leptin, 10 × 10⁻⁹m agonist antibody at 37 °C, 5% CO₂ for 30 min. Cells were next collected and washed once with ice‐cold PBS containing Halt protease and a phosphatase inhibitor mixture (Pierce). The resulting cells were lysed on ice for 30 min with a RIPA lysis buffer (Amresco) containing Halt protease and phosphatase inhibitor mixture. Cell debris was removed by centrifugation at 14 000 × g for 10 min. The amount of total STAT3 and phosphorylated STAT3 was determined by western‐blot analysis of the cell lysates supernatant using anti‐STAT3 antibody (abcam) and anti‐pSTAT3 (Tyr705) (abcam), respectively.

Chromatin immunoprecipitation analysis was performed with sonication method. The purified chromatin from K562/G01 cells was immunoprecipitated with anti-p-STAT3 antibody (Abcam). Anti-STAT3 antibody (Abcam), immunoglobulin G isotype and H2O were used as control. The eluted DNA was purified, and used as PCR template to amplify the promoter sequences between nt −670 and −467 containing putative p-STAT3-binding site at nt −633 to −625 and nt −486 to −478 of RPS27a promoter with the following primers: 5′-GAGCGAAATTCCGTCTC-3′ (forward) and 5′-TGACATTCAGCCTCTGC-3′ (reverse). The PCR products were 184 bp.