Proteasomal degradation of Hax-1. A and B. H1299 cells were transiently transfected with EGFP-Hax-1. Forty-eight hours later, cells were treated with MG132 (1 μM) for 3 hours (A) or Bafilomycin A1 (10 nM) for 18 hours (B). Cells were then harvested and immunoblotted using anti-GFP, ubiquitin, LC3 or GAPDH antibody. Detections of polyubiquitin (A, left, lower panel) and LC3-II (B, left, lower panel) levels in input were used to indicate polyubiquitination levels (A) and autophagy levels (B). Data from three independent experiments were quantified (means ± S.E.M., *p < 0.05, one-way ANOVA). C. Time dependent increase of Hax-1 levels by the proteasomal inhibition (Upper panel). Increased levels of total ubiquitin were served as positive controls for MG132 treatment. Tubulin was served as loading control. D. H1299 cells were treated with CHX with or without MG132 for chase experiments.