Kir3.1 and Kir3.2 expression was performed on the same blot; Kir3.1 blots were re-probed with Kir3.2. The blots were washed in 1X TBS-T (3 times × 10 min) followed by incubation with secondary antibody goat anti-rabbit IgG-HRP conjugate (1:10′000, Jackson Immunoresearch Laboratories Inc, Cat# 111-035-144, RRID: AB_2307391) for 1 h at room temperature. Mouse monoclonal anti-β-actin primary antibody (1:5000, Sigma-Aldrich, Cat# A5441, RRID: AB_476744) and goat anti-mouse secondary antibody (1:5000, Jackson Immunoresearch Laboratories Inc, Cat# 115-035-146, RRID: AB_2307392) were used to measure levels of β-actin. The blots were washed in 1X TBS-T (3 times × 10 min) and then developed with Clarity Western ECL Substrate (Bio-Rad, Cat#170-5061) and scanned with imaging system (Molecular Imager ChemiDoc XRS+, Bio-Rad, USA). Because there was no overlap of immunoreactivity for the Kir3.1, Kir3.2, and β-actin antibodies, levels of these proteins were measured using the same membranes without stripping and reblotting. The images were analyzed with ImageJ (NIH, USA).
