mTOR signaling is activated in Sel1L KO HSCs and Rheb is a novel substrate of Sel1L/Hrd1 ERAD. (A) Representative (left) western blot and (right) summary of quantitation from 3 independent experiments using Mx1-cre⁺; Sel1L^fl/fl (fl/fl) or control (^+/+) mice treated with vehicle or rapamycin. The mice were injected with total 6 doses of pIpC (every 2 days) and injected with rapamycin (4 mg/kg) or vehicle daily (started 6 days before pIpC and continued until the time of analysis). Two weeks after the last dose of pIpC, LSK cells were sorted from the mice for western blot. The levels of mRNA (B, by RT-qPCR; n = 5) and protein level (C, by western blot) of Rheb in HSPCs isolated from Mx1-cre⁺; Sel1L^fl/fl (fl/fl) or control (^+/+) mice were analyzed 2 weeks after pIpC injection. (D-E) The mRNA and protein levels of Rheb in HSCs freshly isolated or cultured in SF-03 media for 24 hours. (F-H) HEK 293T cells were transfected with constructs that overexpress tagged Sel1L, Hrd1, Rheb, or NHK proteins (indicated on top of each blot), and the protein interaction was analyzed by coimmunoprecipitation followed by western blot with antibodies labeled on the right of each blot. (I) HEK 293T cells were transfected with constructs that overexpress HA-tagged ubiquitin, FLAG-tagged Rheb, and Myc-tagged Hrd1, and Rheb proteins were immunoprecipitated by Flag antibody and ubiquitinated Rheb proteins were detected by western blot with HA antibody. At least 3 independent experiments of coimmunoprecipitation or western blots were performed with similar results for each assay. For bar graphs, data represent mean ± SD. The number of replicates (n) indicates independent experiments. Two-sided Student t test was used for statistical analysis.