Figure 5.
Validation of human NSF as an ARIH1 substrate.A, western blot on HEK293 whole cell extracts. HEK293 cells were cotransfected with FLAG-tagged ubiquitin, GFP-NSF, and either FLAG-tagged ARIH1WT (WT) or the catalytically inactive ARIH1C 357S (CI) E3 ligase. GFP-NSF levels were detected with anti-GFP antibody. FLAG-ubiquitin and FLAG-ARIH1 levels (arrowhead in the upper panel) were detected with anti-FLAG antibody. Tubulin was used as loading control. B, GFP-NSF is ubiquitinated by ARIH1. Western blot performed on isolated GFP-tagged NSF in the presence of wild-type ARIH1 (WT) or a catalytically inactive ARIH1 (CI). Ubiquitinated fraction (red) was monitored with anti-FLAG antibody, while anti-GFP was used to detect the nonmodified fraction (green). Below, a quantification of GFP-tagged NSF from three independent pulldowns is shown. Semiquantification of dual-color western blots was performed with Image Lab software (Bio-Rad) and statistical significance determined by two-tailed Student's t test. ∗p-value = 0.0177.