FIGURE 1.

Huwe1 interacts with and ubiquitylates Atoh1. A, following treatment with MG132 (10 μm) in the presence of cycloheximide (CHX; 100 μg/ml), lysates of FLAG-HA-Atoh1 293T cells were processed for Western blotting with FLAG antibody (Atoh1) or β-actin antibody (loading control). Labels indicate the time of chase. DMSO was a vehicle control. B, values for the level of Atoh1 determined by densitometry were plotted to determine half-life. Error bars indicate S.E. Data from three experiments are shown. C, FLAG-HA-Atoh1 293T cells were treated with MG132 (10 μm) or DMSO (Control) for 6 h, and the level of protein was measured both before (5% input) and after (IP: Atoh1) immunoprecipitation. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted (IB) with antibody to ubiquitin. β-actin is a loading control. D, HEK 293T cells were co-transfected with FLAG-Atoh1 and either wild-type HA-ubiquitin (WT), HA-ubiquitin with all lysines except Lys-48 mutated, or empty vector (Control). FLAG-Atoh1 was immunoprecipitated and blotted with antibodies against HA (ubiquitin) and FLAG (Atoh1). FLAG antibody was used to confirm the immunoprecipitation of Atoh1 (lower panel). E, FLAG-HA tagged Atoh1 was purified from whole cell extracts of FLAG-HA-Atoh1 293T cells. Associated proteins were detected by Coomassie blue staining. The areas indicated by the arrows were excised for mass spectrometry (Tables 2 and 3) and Western blotting. F, FLAG-HA-Atoh1 293T lysates were immunoprecipitated with the indicated antibodies (IgG and HA) and subjected to immunoblotting with an antibodies to Huwe1 and HA. G, FLAG-HA-Atoh1 293T cell lysates were subjected to immunoprecipitation using IgG or Huwe1 antibodies followed by immunoblotting with antibodies to HA and Huwe1. H, Atoh1-Huwe1 interaction was assessed by in situ proximity ligation assay (Duolink) in HEK cells transfected with Atoh1 and Huwe1 together or independently. The scale bar is 10 μm.