Ecl supersignal west pico chemiluminescent substrate detection system

PKC epsilon, SOD2, Nrf-2, and Myogenin protein content was tested with a Western blot. In brief, C2C12 was lysed with a RIPA buffer and total protein was quantified with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the instruction protocol. Forty micrograms of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with a specific primary antibody diluted in the blotting solution as recommended by the manufacturer protocol. We used anti-PKCe (Merck Millipore, Darmstadt, Germany, Cat. No.: 06-991), anti-SOD2 (Enzo Life Sciences Inc., Farmingdale, NY, USA, Cat. No.: ADI-SOD-111F), anti-Myogenin (Santa Cruz, Dallas, TE, USA, Cat. No.: sc-12732), anti-Nrf2 (clone: D179C, Cell Signaling, Danvers, MA, USA, Cat. No.: 12721), and anti-HSP70 (Sigma-Aldrich, St. Louis, MO, USA, Cat. No.: H5147) as a loading control. The nitrocellulose membranes were then washed and incubated with 1:5000 peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA) or 1:2000 peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). Proteins were resolved with an ECL SuperSignal West Pico Chemiluminescent Substrate Detection System (Thermo Fisher Scientific, Waltham, MA, USA) and densitometric analyses were performed using ImageJ software (by NIH).

Total proteins were extracted from purified PLT. Briefly, 4.5 × 10⁵ PLT were suspended in a cell lysis buffer supplemented with fresh protease/phosphatase inhibitors and protein concentration was determined. Proteins from PLT and HEK293, as positive control, were separated in 5% SDS-acrylamide gels and blotted onto nitrocellulose membranes. Membranes were blocked and incubated overnight at 4 °C with 1:1000 diluted rabbit polyclonal anti-SGLT2 (Cell Signaling Technologies, Danvers, MA, USA). Mouse mAb anti-GAPDH (1:5000 dilution—Millipore, Burlington, MA, USA) was used as loading control. Membranes were then incubated with 1:2000 peroxidase-conjugated anti-mouse (Sigma Aldrich) secondary antibody and detected by chemiluminescence using ECL Supersignal West Pico Chemiluminescent Substrate detection system (Thermo Scientific, Waltham, MA, USA). NIH ImageJ software was used to scan images and quantify protein expressions.