A mutant U_L38 allele (T23A/Q24A) that shows reduced binding to TSC2 fails to activate metabolic flux.

(A, C, D & E) Confluent MRC5 cells expressing an empty vector control, mutant UL38 T23A/Q24A (mUL38) or WT UL38 (UL38) were cultured in serum free media for 24h, after which conditioned medium and cells were harvested for analysis. (A) Western blot analysis of EV, mUL38 and UL38 cells. (B) 293T cells were transfected with expressing vectors for UL38, mUL38 or FLAG-TSC2 (TSC2-F) proteins, harvested 48h later and immunoprecipitated with a Flag-specific antibody. Lysate (Lys) represents 10% of the IP input. Protein band intensities are shown relative to the FLAG-TSC2+UL38 value. (C-E) Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (n = 3). (F) Confluent MRC5 cells expressing EV, mUL38 or UL38 protein were cultured in serum free media for 24h. Cells were then quenched and extracted for LC-MS/MS analysis. Values are means ± SE (n = 9, except n-carb-asp, where n = 3). (*pL38 or UL38 protein were cultured in serum free media containing DMSO (DMSO) or 100 nm of rapamycin (RAP) for 24h and harvested for western blot analysis.