Fig. 4: MGCG binds to hnRNPK and promotes ATG2A translation in GBM.

A Silver staining of RNA pulldown from GBM cells expressing the biotin-tagged negative and MGCG probes. B Relative abundance of the proteins bound to MGCG determined by mass spectrometry. C RNA immunoprecipitation (RIP) with an anti-hnRNPK antibody in GBM cells. Left: MGCG enrichment was detected by nucleic acid gel electrophoresis. Right: Statistical evaluation of the abundance of IgG and MGCG assayed as shown in the left panel. D FISH and immunofluorescence (IF) were performed to show that MGCG colocalizes with hnRNPK in LN229 cells. E Western blot analysis of the protein levels of hnRNPK after MGCG overexpression or knockdown in LN229 cells. F RT‒qPCR analysis of the relative expression of ATG2A after hnRNPK knockdown in LN229 cells. G Western blot analysis of the protein levels of ATG2A after hnRNPK knockdown in LN229 cells. H RIP was performed with an anti-hnRNPK antibody in GBM cells, and the enrichment of ATG2A was detected by RT‒qPCR. I RNA pulldown was performed using GBM cells expressing the biotin-tagged negative and ATG2A probes, and the expression of hnRNPK was detected by western blotting. J Immunoprecipitation (IP) was performed using LN229 cells with an anti-EIF4B antibody, and the expression of hnRNPK was detected by western blotting. K Far western blot was used to detect the interaction between hnRNPK and EIF4B with EIF4B and hnRNPK recombinant protein. L Western blot analysis of the protein levels of ATG2A after EIF4B knockdown in LN229 cells. M RT‒qPCR analysis of the relative expression of ATG2A after EIF4B knockdown in LN229 cells. N Western blot analysis of the expression of hnRNPK with MGCG overexpression or overexpression of MGCG and simultaneous knockdown of hnRNPK in LN229 cells. Error bars, SEM from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student’s test.