Figure 3. Total and cell-surface protein expression levels of K_V2.1 variants.

A. Representative dot-plots of flow cytometry analyses of CHO cells expressing K_V2.1 tagged with HA-epitope at the extracellular site between S1-S2 linker regions; anti-HA-Alexa488 and anti-K_V2.1-Alexa647 antibodies were used to probe the cell-surface and intracellular K_V2.1 respectively. Population in turquoise (HA⁺/K_V2.1⁺) denote K_V2.1 located at the cell-surface, while the dots in red (HA⁺/K_V2.1^-) indicate expressed K_V2.1 protein located intracellular. WT and non-transfected CHO cells (negative control) are shown at the top left for comparison. Non-transfected CHO cells underwent the entire immunocytochemistry labeling protocol. B. Expression level comparisons of K_V2.1 variants to that of WT (white bars) revealed reduced cell-surface trafficking of variants (* denote variants with intact cell-surface trafficking, i.e. S/T ratio > 0.3): cell-surface expression in turquoise and the total expression in red. C. Correlational analyses of surface-to-total expression level ratio and homomeric I_peak density (values in % WT) result in R²=0.41 and p=0.006: Pearson’s. D. Analyses of two truncation variants, devoid of the epitope site for anti-K_V2.1-Alexa647, further validate the assay. The Y274fsX36 variant fails to reach the cell-surface. In contrast, for the Y533X, the population in green (HA⁺/K_V2.1^-) denote K_V2.1 expressed and reaching the cell-surface, yet unable to be detected by the anti-K_V2.1-Alexa647 antibody due to the absent epitope. E-F. Cross-validation analyses of select K_V2.1variants for cell-surface and total protein expression; Transferrin receptor (TfR), calnexin (CNX). G. Quantification of western blot analyses confirms similar expression pattern as ICC-FC assay.