Figure 1. Localization and differential protein modification of KCNC3^WT, KCNC3^F448L, and KCNC3^R420H in COS-1 and SH-SY5Y cells.

(A) OS-1 cells transiently transfected with either KCNC3^WT or KCNC3^R420H were analyzed by confocal fluorescence microscopy using a KCNC3 antibody. KCNC3^WT shows perinuclear distribution and punctate staining of plasma membrane whereas KCNC3^R420H displays primarily perinuclear staining (white arrows) and very limited fluorescence associated with the plasma membrane. Top and bottom panels were obtained at 40× and 100× magnification, respectively. (B) Immunoblot analysis of KCNC3 in COS-1 cells transiently transfected with human KCNC3^WT, KCNC3^F448L, or KCNC3^R420H expression vectors illustrates a significant reduction of a slower migrating (upper) band consistent with altered posttranslational modifications. (C) Immunoblot analysis of total protein from COS-1 cells transiently transfected with KCNC3^WT or KCNC3^R420H subjected to differential centrifugation verifies that there was no fractional loss of the KCNC3^R420H protein within our isolation protocol and illustrates the diminished upper band. (D) Immunoblot analysis of KCNC3 in stably transfected human neuroblastoma SH-SY5Y cells illustrating the complete absence of the upper band. (E) Immunoblot analysis of KCNC3 in stably transfected SH-SY5Y cells was used to evaluate the treatment of total protein with Endo H or PNGase F. The upper band was resistant to Endo H whereas this species disappeared when total cell extracts were subjected to PNGase F treatment, consistent with the removal of a complex glycan. Both enzymes however led to cleavage of the lower band (control lanes) generating a faster migrating band presumably due to release of a secondary high mannose modification as further illustrated in Figure S1. The removal of a high mannose moiety is consistent with the known activity of Endo H.