Fig. 3: PRELID1P6 interacts with hnRNPH1 and prevents ubiquitination (Ub)-mediated degradation of hnRNPH1.
From: The pseudogene PRELID1P6 promotes glioma progression via the hnHNPH1-Akt/mTOR axis
A Schematic representation of the RNA pull-down assay to identify proteins associated with PRELID1P6. B Mass spectrometry identified the PRELID1P6–protein complex and showed that hnRNPH1 was the major protein in the complex. C RIP assays showed that hnRNPH1 interacted with PRELID1P6. D Nucleotides sites at 141–450 of PRELID1P6 were sufficient to interact with hnRNPH1 protein. FL full-length sequence. E Representative images of costaining PRELID1P6 (red) and hnRNPH1 (green) in U251 and U373 cells by the combination of RNA FISH and immunofluorescence. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. F Knockdown of PRELID1P6 did not decrease the mRNA levels of hnRNPH1 in U251 and U373, as detected by qRT-PCR. G Knockdown of PRELID1P6 decreased the protein expression of hnRNPH1 in glioma cells U251 and U373, as detected by western blotting. H Overexpression of PRELID1P6 increased the protein expression of hnRNPH1 in glioma cells U138 and LNZ308, as detected by western blotting. I U251 and U373 cells with PRELID1P6 knockdown or control cells were treated with MG-132 (5 μM) or vehicle for 24 h. The protein level of hnRNPH1 was increased after MG-132 treatment. J U251 and U373 cells with PRELID1P6 knockdown or control cells were treated with 20 μg/mL of CHX for 0, 15, 30, 60, 120, and 240 min. The protein level of hnRNPH1 levels was analyzed by western blotting. K U251 and U373 cells with PRELID1P6 knockdown. L U138 and LNZ308 with PRELID1P6 overexpression were treated with MG-132 (5 μM) for 24 h. Cell lysates were immunoprecipitated (IP) with either control IgG or antibody against hnRNPH1 and then were analyzed by immunoblotting with a ubiquitin (Ub)-specific antibody. Bottom panel, input of cell lysates. M, N The protein of hnRNPH1 increase in cytoplasm, while decrease in nucleus after PRELID1P6 knockdown in U251 and U373 glioma cells detected by western blot or IF (H3 used as the nucleus control, GAPDH used as the cytoplasm control).
