Rabbit anti cx43 antibody

HeLa cells were lysed in an ice cold lysis buffer (50 mM NaCl, 1% Triton X-100, 50 mM Tris–HCl pH 8.0) containing protease inhibitors (10 μg/mL aprotinin; AppliChem, 10 μg/mL leupeptin; AppliChem and 1 mM PMSF; Sigma Aldrich). Lysates were passed through a 26-gauge syringe needle to shear the genomic DNA and were centrifuged (15 min at 13,000 rpm, 4 °C). For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany). Separation of the immune complex was performed with a magnetic separation unit (Miltenyi Biotec), according to the manufacturer´s instructions. After washing of the columns, the immunoprecipitates were eluted with a Laemmli buffer [51 (link)] and were analyzed for the binding of Cx43 or SHP-2 by Western Blot using a rabbit anti-Cx43 antibody (Sigma Aldrich) or a mouse anti-SHP-2 antibody (Santa Cruz).

Protein samples (25–30 μg of total protein/lane) were mixed 1:1 with 2× protein loading buffer (60 mmol/L Tris‐HCl pH 6.8, 10% glycerol, 2% SDS, 1% bromophenol blue, 10% beta‐mercaptoethanol), boiled 5 min. at 98°C and loaded onto 12% SDS‐PAGE gels. Proteins were detected by Western blotting using: rabbit anti‐Cx43 antibody (C6219; Sigma‐Aldrich), mouse anti‐AIF antibody (MA5‐15880; Pierce‐Thermo Scientific), rabbit anti‐ETFB antibody (17925‐1‐AP; ProteinTech), mouse anti‐Na^+/K⁺ ATPase alpha‐1 antibody (05‐369; Millipore, Billerica, MA, USA), mouse anti‐SDHA (MS204; MitoSciences, Eugene, OR, USA), mouse anti‐Pan Cadherin (C1821; Sigma‐Aldrich) and rabbit anti‐TOMM20 antibody (HPA011562; Sigma‐Aldrich).