Frozen aliquots of CHO-K1 cells electroporated with WT or variant KCNB1 channels were thawed and grown under the same conditions used for automated patch clamp recording. Cell-surface biotinylation and immunoblotting were performed as previously described7 (link),14 (link), 60–72h post-electroporation, using mouse anti-KV2.1 (1:250; K89/34, Antibodies Inc), mouse anti-transferrin receptor (1:500; H68.4, ThermoFisher), and rabbit anti-calnexin (1:250; H70, Santa Cruz Biotechnology), Alexa Fluor 680-goat anti-rabbit IgG (1:20,000, Jackson ImmunoResearch) and Alexa Fluor 790-goat anti-mouse IgG (1:20,000, Jackson ImmunoResearch) antibodies.
Normalized total, surface, and surface/total protein ratio results were derived from 3 independent experiments. Calnexin immunoreactivity was present in total protein lysates and absent from the cell-surface fraction, confirming the selectivity of biotin labeling for cell-surface protein.
Normalized total, surface, and surface/total protein ratio results were derived from 3 independent experiments. Calnexin immunoreactivity was present in total protein lysates and absent from the cell-surface fraction, confirming the selectivity of biotin labeling for cell-surface protein.