Figure 7.

αkap had no effect on the stability of AChRα when lysines essential for ubiquitination were mutated. HEK293T cells were cotransfected with plasmids encoding the indicated AChRα variants and HA–Ub, and 48 h later protein lysates were collected. The anti-AChRα antibody MAB153 was used to selectively immunoprecipitate proteins from each sample. A, The extent of ubiquitination of AChRα detected with an anti-HA antibody. In this experiment, the amount of material loaded onto each lane was adjusted so that comparable amounts of AChRα were present in all samples. The top gel shows the amount of AChRα detected with anti-AChRα (MAB210), and the bottom gel shows the anti-HA immunoreactivity. The control lane (Cont) was from cells transfected with HA–Ub only. WT, Wild type. The 1R lane is the K359R mutant, the 3R lane is the K359R/K388R/K393R mutant, and the 4R lane is the K359R/K374R/K388R/K393R mutant. The lower band present in all lanes of the anti-HA blot (arrow) is the 25 kDa light chain of the antibody used for immunoprecipitation and served as a loading control. The smear present in some lanes (brackets, ∼70–250 kDa) is polyubiquinated AChRα and so is absent in the control because no AChR were present. Polyubiquitination of AChRα was greatly decreased in the 3R and 4R mutants. B, Whole-cell recordings from HEK293T cells cotransfected with EGFP, wild-type AChR β, δ, and ε subunits, and either the wild-type or the 4R mutant AChRα. The holding potential was −50 mV. ACh was applied for the duration indicated by the bar above each trace. C, Western blots of homogenates from HEK293T cells cotransfected with EGFP and wild-type AChRα or the ubiquitination-deficient 3R or 4R mutant. In these experiments, the same amount of material (as determined from anti-GFP staining) was loaded onto each lane. No αkap was present (−αkap). D, Quantification of three independent experiments similar to C. In this panel as well as F, 100% represents the intensity of signal produced when cells were transfected with wild-type AChRα in the absence of αkap. E, Experiment similar to C, except that the cells were also cotransfected with a plasmid encoding αkap (+ αkap). F, Quantification of three experiments similar to E.