Monoclonal Anti-Connexin-43 antibody produced in mouse

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	-Mouse (1:8000)

Protocol tips
-Mouse (1:8000)

Publication protocol

Hearts from WT, mdx, and mdx:utr mice were dissected out, rinsed in sterile PBS, and flash frozen in liquid nitrogen. Mouse tissues were homogenized in M-PER Mammalian Protein Extraction Reagent (Pierce) supplemented with EDTA, PMSF, NaVO3, Okadaic Acid, NaF, Benzamidine, and NaPyr. Human tissues were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P40, 0.5% Sodium Deoxycholate, 0.1% SDS, 5% glycerol) supplemented with protease and phosphatase inhibitor cocktails (Roche, cOmplete ULTRA Mini and PhosSTOP). Western blots performed without the use of proper phosphatase inhibitors yield a single band for Cx43 migrating near 43 kda, rather than separate np-Cx43 and p-Cx43 bands. Protein concentrations were estimated using a BCA kit (Pierce). 5 μg of protein per sample was separated on 12% gels (Bio-Rad) and run under the same experimental conditions, transferred to PVDF or nitrocellulose membranes, and probed with the following antibodies: Cx43 (Sigma, C8093, 1:8000), GAPDH (Cell Signaling, 3683, 1:2000), Panx1 (Santa Cruz, sc49695, 1:1000) or Nav1.5 (Sigma, S0819, 1:500). Blots were developed using enhanced chemiluminescence (ECL) and densitometric analysis was performed using Fuji Images or Bio-Rad Image Lab software.

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