Figure 2.

KLHL41-NRAP interaction results in ubiquitination and proteasome-mediated degradation of NRAP. (A) Co-immunoprecipitation in C2C12 cells transfected with KLHL41-FLAG and NRAP-V5 demonstrating interaction of the two proteins in the myogenic context. (B) Overexpression of KLHL41-FLAG and NRAP-V5 in the presence of ubiquitin-HA in C2C12 results in increased ubiquitination and decreased stability of NRAP. (C) Western blot analysis, quantification of NRAP mRNA and protein in control or Klhl41 knockout myotubes treated with MG132. (D) Western blot analysis and quantification of NRAP in control or Klhl41 knockout myoblasts treated with cycloheximide for different time intervals. The NRAP band intensity was normalized to GAPDH and then normalized to t = 0 hr controls (bottom panel). Data are shown as mean ± s.d. and blots are representative of three different experiments (n = 3). P-values were calculated using a two-tailed Mann–Whitney U-test, ^*P < 0.01.