Enhanced chemiluminescence ecl reagent

Total protein was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Proteins were separated using 12% SDS‐PAGE, and were then transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the PVDF membranes were blocked by utilizing Tween 20 (TBST) and Tris‐buffer saline that contained 5% skimmed milk powder at room temperature for 1.5 hours. The membranes were incubated with specific rabbit‐anti‐human PTEN monoclonal antibodies (1:1000, Abcam, Cambridge, MA, USA), rabbit anti‐human monoclonal antibodies for E‐cadherin, vimentin and N‐cadherin (1:1000, Proteintech, Manchester, UK) and rabbit anti‐human GAPDH monoclonal antibodies (1:1000, CST, Danvers, MA, USA) at 4°C for all night. After irrigating the membranes with TBST buffer for 3 times, and peroxidase‐labelled mouse anti‐rabbit secondary antibody (Zhongshan Bio‐Engineering, Guangzhou, China) were supplemented to the membrane for 2 hours incubation at room temperature. Lastly, enhanced chemiluminescence (ECL) reagents (Bio‐Rad, USA) were employed to make proteins visible, and Image J software (National Institutes of Health, Bethesda, MD, USA) was arranged to semi‐quantify the proteins.

Ang III and Ang II were purchased from Bachem (Torrance, CA, USA). Tissue culture supplies, such as fetal bovine serum (FBS), Dulbecco’s modified Eagles Medium (DMEM)/F12 (1:1), streptomycin, penicillin and trypsin/ethylenediamine tetraacetic acid (EDTA) were purchased from Fisher Scientific (Milford, MA, USA) or VWR (Suwannee, GA, USA). The AT₁R blocker (Losartan) was provided by Du Pont Merck (Wilmington, DE, USA). The AT₂R blocker (PD123319), and the JAK2 inhibitor (AG490) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein measurement supplies, acrylamide, enhanced chemiluminescence (ECL) reagents, nitrocellulose membrane, gel electrophoresis and Western blotting supplies inclusive of bicinchoninic acid (BCA) protein reagents were purchased from either Bio-Rad Laboratories (Hercules, CA, USA) or VWR (Piscataway, NJ, USA). The non-phosphorylated and phospho-specific STAT3 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Applied Biosystems (Foster City, CA, USA) supplied reverse transcription reagent kit and TaqMan gene expression primers for rat IL-6. An ELISA kit for IL-6 was purchased from R&D Systems (Minneapolis, MN, USA). All the other chemicals were obtained from either Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Milford MA, USA) or VWR international (Suwannee, GA, USA).

Total protein was obtained from cell pellets previously washed with PBS, through digestion via the lysis buffer (Bio-Rad, Hercules, CA, USA). Samples were loaded on polyacrylamide gels for electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Blocking was performed for 1 h with 5% milk solution, and membranes were incubated with the primary antibody overnight. After washing, the membranes were incubated with the Secondary IgG-HRP secondary antibody (Cell Signaling, MA, USA), ultimately being rinsed and envisioned by using the enhanced chemiluminescence (ECL) reagent (Bio-Rad, Hercules, CA, USA).