Figure 3. The lead compound O03 stabilizes CLRN1^N48K by a post-translational mechanism.

(a) Mouse CLRN1^N48K expressed in NIH/3T3 cells was stabilized by O03 treatment. (b) Quantitative RT-PCR of HEK293 cells stably expressing human CLRN1^N48K. CLRN1 mRNA levels were not affected by either bortezomib or O03 treatment (O03 vs. DMSO, P = 0.9995, two-sided t-test, n=4, means ± SDs). (c) Protein synthesis is not required for the effect of O03. Cells treated with proteasome inhibitors bortezomib and MG132 showed increased levels of CLRN1^N48K as did O03 treatment when compared to DMSO (lanes 1–4, T0). CLRN1^N48K levels remained similar after treatment for an additional 6 h in the presence of 100 μM cycloheximide (CX) (lanes 5–7, T1), indicating that O03 stabilized CLRN1^N48K in the absence of protein synthesis. To confirm that CLRN1^N48K can be effectively degraded in the absence of protein synthesis, cells were treated with bortezomib, MG132, or O03 for 6 h. Compounds then were washed out, and cells were further incubated for 6 h in the presence of CX (lanes 8 – 10, T1). CLRN1^N48K was undetectable in cells after removing the reversible proteasome inhibitor MG132 (compare lanes 3 and 9). Effects of O03 and bortezomib persisted 6 h after the washout. Note: Multiple immunopositive bands were detected by the HA-antibody which recognizes the Ct-tail of CLRN1^N48K (a and c). Based on the analysis of the primary structure³⁹, CLRN1 contains a few signal peptide cleavage sites; incomplete cleavage leads to a few differently sized bands. See also Supplementary Figure 13.