FIGURE 7.

Identification of residues within the RCLRG motif critical for protein degradation by alanine scanning mutagenesis. Shown are Cos-7 cells expressing wild-type KCNQ2, fsQ2–865X, or different forms of the fsQ2–865X mutant in which each residue of the RCLRG motif was mutated to alanine (-ACLRG, -RALRG, -RCARG, -RCLAG, -RCLRA) or two residues were mutated to alanine (-AALRG and -RCLAA). The fsQ2–865X mutant has 864 residues (see Fig. 1) and carries the RCLRG motif before the stop codon as indicated by the letter X. Cells were treated with CHX (75 μg/ml) for 0 or 12 h or with vehicle (0.75 % DMSO) for 12 h. All extracted proteins (left panels for KCNQ2, middle panels for fsQ2–865X and individual mutations, and right panels for double mutations of the fsQ2–865X mutant) were separated by SDS-PAGE and Western-blotted with anti-FLAG or anti-actin antibody.