Figure 4.
SRM promotes the translation of HMGA2 by eIF5a hypusination. A, WT and SRM KO MGH-U3 cells were treated with 100 μmol/L spermidine in serum-free medium as indicated for 24 hours. GAPDH was used as internal control. B, MGH-U3 cells were treated with 10 μmol/L GC7 or PBS for 24 hours. eIF5AHyp and HMGA2 levels were examined by Western blotting (left) and qRT-PCR (right). GAPDH was used as an internal control. C, MGH-U3 cells were treated with 10 μmol/L MCHA or DMSO for 96 hours. eIF5AHyp and HMGA2 levels were examined by Western blotting (left) and qRT-PCR (right). GAPDH was used as an internal control. D, OPP pulldown assay for nascent protein synthesis. OPP is incorporated into newly translated proteins, and then the azide group on biotin is conjugated to an alkyne group on OPP by click reaction. Biotin-labeled proteins are pulled down by magnetic streptavidin beads and detected by Western blotting. E, The expression of newly synthesized HGMA2 protein of indicated MGH-U3 cells treated with 10 μmol/L MCHA or DMSO for 96 hours using the method shown in D. F, The mRNA from fractions taken during polyribosome profiling of WT and SRM KO MGH-U3 cells were isolated and quantified for HMGA2. Actb, β-actin. G, Schematic of HMGA2-mCherry WT and mutant (MUT) constructs. H, Western blotting with the indicated antibodies in WT and SRM KO MGH-U3 cells transfected with indicated HMGA2-mCherry constructs from G. GAPDH was used as an internal control. Data are presented as the means ± SD from three independent experiments. ns, nonsignificant (Student t test). ns, nonsignificant.