Anti actin clone ac 40

3×100mm dishes of HEK293 cells were transfected with 2.5μg DNA for each separate construct/dish. Cells were collected 48h post-transfection, washed three times with PBS. Biotinylation was performed in 10 mL ice-cold PBS containing 0.25 mg/mL sulf-NHS-SS-Biotin for 30 minutes at 4°C. 10mM glycine was added to quench the reaction. Cell lysis solution contained (in mM/L) 50 HEPES (pH 7.4), 150 NaCl, 1.4 MgCl2, 1 EGTA, 10% Glycerol, 1% triton X-100, 1.2mg/mL N-Ethyl-maleimide with protease inhibitors. NeutrAvidin Agarose was used to pull down labeled proteins. Eluted proteins were then used for Western blotting as previously described,^{11 (link)} and blotted using a sodium channel antibody (Millipore Polyclonal Anti-Na+ Channel III–IV loop). Pan-cadherin (Cell Signaling Technology) was used as a loading control for the cell surface biotinylated fraction and actin (Sigma-Aldrich monoclonal Anti-Actin, Clone AC-40) was used as a negative control for the cell surface biotinylated fraction. To determine the protein expression level, the sodium channel bands were normalized to the control bands (actin for total lysate and Pan-cadherin for biotinylated fractions).