Fig. 3: Pdcd4 selectively represses the translation of BDNF splice variant IIc mRNA in an eIF4A-dependent manner.

a Mice hippocampal lysates were subjected to RNA-IP with Pdcd4 antibodies or IgG. Purified RNA was analyzed by RT- PCR using specific primer for BDNF. b Luciferase assays were performed after transfecting each pGL3 construct into HEK 293 cells transfected with either siNC or siPdcd4. Renilla luciferase vector was co-transfected for normalization. Three independent experiments are shown, mean ± SEM, unpaired two-tailed Student’s t test, *P < 0.05. c Luciferase assays were performed after transfecting each pGL3 construct into HEK 293 cells transfected with either pcDNA3.1 or Flag-Pdcd4. Renilla luciferase vector was co-transfected for normalization. Three independent experiments are shown, mean ± SEM, unpaired two-tailed Student’s t test, *P < 0.05. d GFP antibody detected BDNF expression after transfecting each BDNF-GFP construct into HEK 293 cells with either siNC or siPdcd4. e GFP antibody detected BDNF expression after transfecting different dose Pdcd4 plasmid with each BDNF-GFP construction. f Human IIc-isoform BDNF 5’ UTR secondary structure was predicted in the website: http://rna.tbi.univie.ac.at/, and based on principle of the minimum free energy and base pair probabilities from single RNA sequences. g Western blot was performed after transfecting each IIc-5’UTR mutation of BDNF-GFP construct into HEK 293 cells transfected with either pcDNA3.1 or Flag-Pdcd4. h Luciferase assays were performed after transfecting both pGL3-BDNF variant IIc-5’UTR and siPdcd4 constructions into HEK 293 cells transfected with Pdcd4 mutations. Renilla luciferase vector was co-transfected for normalization. Three independent experiments are shown, mean ± SEM, unpaired two-tailed Student’s t test. *P < 0.05. i IIc-5’UTR-BDNF-GFP construction with Pdcd4 mutations were transfected into HEK 293 cells, and GFP antibody detected BDNF expression.