HuD-A inhibits insulin translation.

<p><i>In vitro</i> translation assay of chimeric Ins2L-5’UTR-Luc-Ins2-3‘UTR <b>(A)</b> in presence of Long/Short (L/S) unlabeled RNA along with His-PABP <b>(B)</b> in presence of His-GFP or His-HuD A/B and D. Lower panel indicates the amount of luciferase and renilla RNA at the end of translation reaction detected by RT-PCR. Renilla was used as internal control for translation efficiency and His-GFP was used as nonspecific protein control and <b>(C)</b> in presence of recombinant PABP and/or HuD along with nonspecific protein (NSP). In all three experiments, the Luc/Renilla ratio was normalized and relative luciferase activity is expressed as a percentage of control and represented as bar graph. <b>(D)</b> MIN6 cells were transfected with HA-tagged HuD plasmid along with Long-5’UTR-Luc-Ins 3’UTR and Renilla luciferase plasmid. After 16 hours post transfection, the cells were treated with low glucose (2 mM) or high glucose (20 mM) for one hour and luciferase activity was measured. HuD expression was assessed by western blot analysis of the lysate (lower panel) <b>(E)</b> Secreted insulin levels in the low glucose condition media was assayed by ELISA in MIN6 cells overexpressing different HuD isoforms. The insulin readings were normalized to the control cells transfected with only empty vector (EV). <b>(F)</b> Intracellular proinsulin levels in low glucose condition were assayed in MIN6 cells by western blot analysis after overexpressing HA-tagged HuD isoforms along with empty vector control. HuD expression was assessed by Western blot analysis (lower panel). The graph represented in A, B, C and D are average of three experiments and the error bar represents the ± SEM and * indicates p<0.05 when compared with control.