Fig. 3.

B31 tolerance to high K⁺ represents the activity of trafficking signals that down-regulate surface expression of membrane proteins. (A) Sequences that were fused to the C-terminus of Kir2.1 channels. (B) Total expression levels of Kir2.1 fusions. Total lysates from HEK293 cells transfected with HA-Kir2.1 constructs were resolved by SDS–PAGE and immunoblotted for HA. (C) Association of COPI with Kir2.1 fusions. The Kir2.1 fusions immunoprecipitated with HA Ab were resolved by SDS–PAGE and immunoblotted for HA (lower panel) and the associating β-COP (upper panel). (D) Surface expression of Kir2.1 fusions. HEK293 cells were transfected with HA-Kir2.1 constructs and analyzed for cell surface expression by FCM. The histograms (left panels) of Kir2.1-transfected cells (filled) were overlaid with that of vector-transfected cells (unfilled). The Median values were determined for the total cell populations and shown in average±s.d. of triplicate samples from the representative of three experiments (right panel). (E) Growth assay of B31 expressing Kir2.1 fusions. The Kir2.1- or pYES2met vector-transformed B31 cells were plated on YNB media (pH 6.50) with indicated concentrations of KCl and cultured at 30 °C. The images were photographed at Day 7. (F) Expression of Kir2.1 channels in B31 cells. The proteins were extracted from the B31 cells transformed with pYES2met vector or the indicated Kir2.1 constructs. The samples were resolved by SDS–PAGE and immunoblotted for Kir2.1 (upper panel). The lower panel shows the Ponceau S staining of the transfer membrane, indicating similar loading of the proteins for each sample.