95°C for 5 min, and 16 μL (1.25 mg/mL) of total protein per lane was loaded onto 12.5% Tris-Glycine gel. Gel electrophoresis was performed at 100 V for 2 h with 10% Tris-Glycine running buffer (100 μL 10X Tris-Glycine, 10 μL 10% SDS, and 890 μL filtered deionized H2O). Proteins were transferred to a low-fluorescent, hydrophobic polyvinylidene difluoride (PVDF) membrane at 95 V for 1 h in the cold room with 0.45 μm pore size (Immobilon-P, EMD Millipore, MA) in 10% methanol transfer buffer (100 μL MeOH, 100 μL 10X Tris-Glycine, and 800 μL filtered deionized H2O) and the membranes were blocked with 5% nonfat milk in TBS-T solution (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with following primary antibodies: rabbit polyclonal anti-Kir3.1 (1:1′000, Alomone Labs, Cat#APC-005), rabbit polyclonal anti-Kir3.2 (1:1′000, Alomone Labs, Cat#APC-006), or rabbit polyclonal anti-Dyrk1A (1:1′000, Cell signaling, Cat#2771S). Comparison of 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
