Fig. 3. PRRT2 does not affect NKA expression level at the membrane surface.

A, B Left: Representative immunoblots of cell surface biotinylation performed in WT and PRRT2 KO hippocampal neurons. Total lysates (TOTAL), biotinylated (cell surface, EXTRA), and non-biotinylated (intracellular, INTRA) fractions were analyzed by western blotting with antibodies to PRRT2 and either α3-NKA (A) or α1-NKA (B). Antibodies to transferrin receptor (Transf Rec) and actin were used as markers of cell surface and intracellular fractions, respectively. Vertical lines in the blot indicate that the lanes were on the same gel but have been repositioned in the figure. Right: Total and cell surface α3-NKA (A) and α1-NKA (B) immunoreactivities are expressed in percent of the respective WT value after normalization to Transferrin receptor (for the EXTRA fraction) or actin (for the INTRA fraction). Means ± SEM of n = 3 independent experiments; unpaired Mann–Whitney’s U-test. C, D Left: Representative images of a WT neuron transfected with α3-NKA-SEP (C) or α1-NKA-SEP (D) perfused with Tyrode followed by MES buffers (scale bar, 20 μm). Insets show higher magnification of the fluorescence at the plasma membrane where ROIs were measured. The cartoons shown on top depict the SEP state under Tyrode and MES buffers, respectively. The green stars represent fluorescent emission of the chimeric protein, which is quenched at acidic pH, i.e., in acidic intracellular compartments and in the presence of extracellular MES buffer. Right: Membrane expression of α3-NKA-SEP (C) and α1-NKA-SEP (D) in WT and PRRT2 KO neurons, calculated as the normalized difference between fluorescence in Tyrode (F₀) and fluorescence in MES (F_MES) as [(F₀−F_MES)/F_MES]. Data are means ± SEM of 40–51 neurons, obtained from n = 3 independent preparations for WT and PRRT2 KO, respectively.