Lds gel loading buffer

Co-immunoprecipitated fractions taken from solubilized cell lysate (L), unbound waste (U), bead wash (W), and final elution (E) were mixed with LDS gel loading buffer (Thermo-Fisher, #NP0007) containing 50mM fresh dithiothreitol and heated to 65°C for 5 minutes. Samples were then run on NuPage 4–12% Bis-Tris Protein Gels (Thermo-Fisher, #NP0322) according to manufacturer’s instructions. Samples were then transferred to Immobilon PVDF membranes for Western blotting (Sigma-Aldrich, #IPSN07852) according to manufacturer’s instructions. Protein-adhered membranes were blocked with TBS-T (50mM Tris, 150mM NaCl, 0.1% Tween 20) with 3% BSA for 1 hr followed by overnight incubation with primary antibodies to detect mVenus (Anti- GFP, rabbit, 1:1000 dilution, Novus Biologicals, #NB600–308) and FLAG (Anti-FLAG- M2, mouse, 1:1000, Sigma-Aldrich, F1804) at 4°C. The following day membranes were washed 4 × 15 min in TBST and probed for 1 hr with anti-rabbit IgG HRP (1:5000, Jackson ImmunoResearch, #711-035-152) for mVenus detection or anti-mouse IgG HRP (1:3000, Cell Signaling, #7076S) for FLAG detection. Blots were washed again 4× 15 min in TBST, mixed with Clarity Western ECL Substrate (BioRad, #1705061) and imaged on a ChemiDoc Touch Imaging System (BioRad, #1708370).

We homogenized ∼5 mm-long mouse mesenteric artery tissue samples (1 sample each from 4-5 different mice per sex per genotype) in 100 μl of PBS (pH 7.5) containing protease inhibitor cocktail (Thermo Fisher, Waltham, MA, USA) and 10% SDS (w/v) using a motorized Eppendorf homogenizer. Samples were then centrifuged for 10 minutes at 3 × g at room temperature (to avoid solidification arising from the high SDS content). The supernatants were resuspended in LDS gel loading buffer (Thermo Fisher) containing 25 mM tris(2-carboxyethyl)phosphine, heated for 10 minutes at 65 °C, vortexed, centrifuged for 3 minutes at 5 × g, and then separated by SDS-PAGE. Proteins were transferred to PVDF membranes and western blotted with 1/500 rabbit polyclonal anti-KCNQ4 (Santa Cruz Biotechnology, Dallas, TX, USA) and 1/1000 rabbit polyclonal anti-GAPDH (Abcam, Cambridge, UK) antibodies, with chemiluminescent detection via 1/5000 HRP-conjugated goat anti-rabbit IgG secondary antibodies (BioRad, Hercules, CA, USA). Band densities were quantified using ImageJ software (NIH, Bethesda, MA, USA) and Kv7.4 band density values each normalized to same-lane GAPDH band density.