P stat3 727

Skin fibroblasts from a healthy donor were cultured with 10 ng/ml of IL-6 and metformin for 1 h. Proteins were isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Western blotting was performed with a SNAP i.d. Protein Detection System (Millipore, Billerica, MA, USA). Membranes were stained with primary antibodies against phosphorylated mTOR (p-mTOR; Ser2448) (D9C2), phosphorylated STAT3 (p-STAT3; 727), and β-actin (all from Cell Signaling Technology, Danvers, MA, USA).

Cells or tumor tissues were collected and suspended in RIPA lysis buffer (Biomiga, Inc.) containing a cocktail of proteinase inhibitors (Roche). The protein concentration was quantified using the bicinchoninic acid (BCA) assay kit (Thermo scientific, Inc.) to ensure that equal amounts of protein from different subpopulations were loaded into the gel. The proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against murine GZMM (P-15, Santa Cruz Biotechnology, Inc.); E-cadherin, N-cadherin, vimentin, AKT, p-AKT, ERK1/2, p-ERK1/2, STAT3, p-STAT3⁷⁰⁵, p-STAT3⁷²⁷ (Cell Signaling Technology), a-tubulin (ZSGB-BIO), and β-actin (ZSGB-BIO) were used at 1:1000 dilution and incubated with the membranes at 4°C overnight. The reaction was revealed by horseradish peroxidase (HRP)-coupled secondary reagents (Pierce, Rockford, IL, USA), developed by enhanced chemiluminescence (ECL) (Applygen Technologies Inc.) according to the manufacturer's instructions, and subjected to exposure using LAS4010 (General Electric Company).

Thoracic aorta was grinded using homogenizer. The aortic homogenation and cultured VSMCs were lysed with 0.25 M Tris (pH 6.8), 1.5% SDS, and 1% mercaptoethanol on ice. The lysis samples were subjected to electrophoretic separation with 10% SDS polyacrylamide gel electrophoresis and transferred onto NC membrane (Millipore, Schwalbach, Germany) as described earlier [29 (link)]. Membranes were pre-incubated with 5% dry-milk powder incubated 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibody Y1R (1:500, Abcam), Y5R (1:500, Abcam), STAT3 (1:500, Cell Signaling), P-STAT3 (705) (1:500, Cell Signaling), P-STAT3(727) (1:500, Cell Signaling), c-Fos (1:500, PTG), goat polyclonal antibody NPY2R (1:500, Sigma), rabbit polyclonel antibody PCNA(1:1000, PTG), mouse polyclonal antibody GAPDH (1:1000, PTG), respectively. After incubation with alkaline phosphatase-conjugated secondary antibodies (Jackson Immunoresearch), the signals were visualized by nitroblue tetrazolium—bromochloroindolyl phosphate (Bio Basic, Inc.) and quantified with Quantity One software (Bio-Rad).