(A) KLHL41 regulation of NEBfrag in the absence or presence of proteasome inhibitor (10 µM MG132 for 24 hr) was determined by western blot analysis with the indicated antibodies in COS-7 cells. Note that DMSO treated cells were used as negative (-) control. Expression of NEBfrag was not detected in the presence of MG132, and KLHL41 stabilizes NEBfrag in the presence or absence of MG132. (B) KLHL41 regulation of NEBfrag in the absence or presence of autophagy inhibitor (10 µM chloroquine for 12 hr) in COS-7 cells. Note that DMSO treated cells were used as negative (-) control. Expression of NEBfrag was not detected in the presence of chloroquine, and KLHL41 stabilizes NEBfrag in the presence or absence of chloroquine. Increased level of LC3 protein was detected as a positive control for autophagy inhibition. (C) Multiple NEBfrag deletion mutants are stable in the absence of KLHL41. COS-7 cells were transfected with various NEBfrag deletion mutants in the absence (top) or presence (bottom) of KLHL41. Protein levels were determined by western blot analysis with the indicated antibodies. Co-immunoprecipitation of FLAG-KLHL41 and NEBfrag deletion mutants (bottom) shows that all mutants interact with KLHL41. (D) Expression of KLHL41 affects NEBfrag solubility. COS-7 cells were transfected with the indicated plasmids and lysed with mild detergent to collect supernatant (SN). The remaining pellet (P) was then solubilized under high detergent conditions. Expression of FLAG-KLHL41 and NEBfrag in SN and P fractions was detected by western blot analysis. In the absence of KLHL41, NEBfrag was exclusively expressed in P. However, in the presence of KLHL41, total NEBfrag levels were increased and enriched in the SN fraction. GAPDH was restricted to the SN fraction. Fibrillarin, a nucleolar protein, was enriched in P. (E) Expression of NEBfrag in the absence and presence of KLHL41 was detected by immunofluorescence of COS-7 cells transfected with the indicated plasmids. Note that NEBfrag was accumulated in the nucleus in the absence of KLHL41 but it was redistributed to the cytoplasm when KLHL41 was co-expressed. Scale bar: 10 µm. (F) Model for regulation of KLHL41 stabilizing activity. KLHL41 acts as a poly-ubiquitin dependent chaperone that prevents NEB aggregation. Loss of KLHL41 or inhibition of its poly-ubiquitination leads to NEB aggregation, sarcomere disarray and nemaline myopathy.