Monoclonal anti ha agarose affinity beads

Naïve Hek293 cells expressing the PRRT2 variants were harvested in lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA and 1% Triton X-100 supplemented with protease inhibitor cocktail) 48 h after transfection and centrifuged at 10,000g for 10 min at 4 °C. Kept an aliquot for input, the supernatant was incubated with 50 μl of monoclonal anti-HA-agarose affinity beads (Sigma-Aldrich) for 2 h at 4 °C. After three washes in lysis buffer, the beads were incubated with cell extracts from Hek293 cells expressing either Nav1.2 or Nav1.1 for 2 h at 4 °C. After extensive washes in lysis buffer and detergent-free lysis buffer, samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subjected to Western blotting with pan-Nav (1:300, Sigma-Aldrich Cat.S8809, RRID:AB_477552) and HA (1:1000, Thermo Fisher Scientific Cat.71–5500, RRID:AB_2533988) specific antibodies. Actin immunoreactivity (anti-actin antibody 1:1000, Sigma-Aldrich Cat.A4700, RRID:AB_476730) was used as control of equal loading.

Wild-type HEK293 cells or Na_V1.2-expressing HEK293 cells were transfected as previously described. After 48 h, cells were harvested in lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA and 1% Triton X-100 supplemented with protease inhibitor cocktail) and centrifuged at 10000 × g for 10 min at 4 °C. Kept an aliquot for the input sample, the supernatant was incubated with 50 μL of either anti-FLAG® M2 Affinity Gel or monoclonal anti-HA-agarose affinity beads (Sigma-Aldrich) at 4 °C for 2 h. After extensive washes in lysis buffer and detergent-free lysis buffer, samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blotting with anti-panNa_V (1:300; Sigma-Aldrich), anti-FLAG (1:2000; Sigma-Aldrich), or anti-HA (1:1000; Thermo Fisher Scientific) specific antibodies.