Anti erk1 2 sc 514302

Media and supplements were from Corning (Tewksbury, MA, USA). Spexin was purchased from Phoenix Pharmaceuticals. Galanin receptor antagonists were purchased from Tocris Bio-techne. Antibodies: anti-myogenin (sc-12732), anti-Myh (sc-376157) and anti-MyoD (sc-377460), anti-vinculin (sc-73614), anti-GALR2 (AF1463a), anti-GALR3 (AP16443), anti-phospho-ERK1/2 (sc-7383) and anti- ERK1/2 (sc-514302) were from Santa Cruz Biotechnology (Dallas, TX, USA) or Abgent (San Diego, CA, USA); anti-βactin A2228 and all secondary antibodies for Western blot were from Sigma-Aldrich (Taufkirchen, Germany). Unless otherwise stated all other reagents were from Sigma-Aldrich.

Total proteins of NHEK cells were extracted with RIPA buffer containing 1 mM DTT and 1 U of EDTA-free protease inhibitor cocktail (Roche, Manheim, Germany). An equal amount of protein was separated on 7.5% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked using 5% skim milk in TBS at 25 °C for 2 h and incubated with specific primary antibodies: anti-GAPDH (2188, Cell Signaling, MA, USA), anti-NF-κB (3035, Cell Signaling), and anti-ERK1/2 (sc-514302, Santa Cruz Biotechnology, CA, USA) at 4 °C overnight. Membranes were incubated with secondary antibodies (HRP-linked IgG) at 25 °C for 1 h. The secondary antibodies were anti-mouse (A90-116P, Bethyl Laboratories, Montgomery, TX) and anti-rabbit (A120-101P, Bethyl Laboratories). Membranes were washed with TBST, and proteins were detected using enhanced chemiluminescence (ECL) Prime Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ, USA). The GAPDH band was used as a loading control.
The supernatants of NHEK cells were collected, and TNF (TNF-alpha Human ELISA Kit, High Sensitivity, BMS223HS) and IL-6 (IL-6 Human ELISA Kit, High Sensitivity, BMS213HS) were measured using commercial ELISA kits (Invitrogen, CA, USA) in accordance with the manufacturer’s protocols.