Mouse monoclonal anti tubulin

Cells were lysed in buffer with 50 mM Tris-HCl pH.8, 150 mM NaCl, 5 mM EDTA, 1% NP-40 (Igepal AC-630). Extracts were centrifuged at 14000 rpm for 10 min to remove cell debris. Protein concentrations were determined by colorimetric assay (Bio-Rad). Western blotting was performed using the following primary antibodies: mouse monoclonal anti-Tubulin (Abcam), rabbit monoclonal anti-p21 (cell signalling). Secondary antibodies used were goat anti-mouse, goat anti-rabbit conjugated to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ, USA). Immunostained bands were detected by chemiluminescent method (Uvitec Alliance, Cambridge).

Protein analysis was performed as previously described with minor modifications (Barajas‐Martinez et al. 2009; Calloe et al. 2010). Ventricular tissue from three dogs was snap frozen in liquid nitrogen and stored in −80°C prior to protein isolation. Membrane proteins were isolated using the Proteo Extract Native Membrane Kit (Calbiochem) according to the manufacturer's protocol. Protein concentration was determined by BCA assay (Pierce BCA Protein Assay). Samples were denatured 10 min at 65°C with 355 mmol/L β‐mercaptoethanol, separated on precast polyacrylamide 4–15% Tris‐HCl gels (BioRad) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4°C with the following primary antibodies. Rabbit polyclonal Anti‐Nav β1‐β4 (1:3000 dilution, Alomone Labs) and mouse monoclonal antitubulin (1:3000, Abcam) were used as a loading control. Secondary antibodies were HRP‐conjugated goat anti‐rabbit IgG (1:10000, BioRad) and goat anti‐mouse IgG (1:10000, Bio‐Rad).