Fig. 4: MIR155HG interacts with YBX1 protein to increase the protein stability.

A Proteins retrieved from the MIR155HG pull-down assay were analyzed by SDS-PAGE. B Western blot analysis of the proteins retrieved from the MIR155HG pull-down assay using an anti-YBX1 antibody. C RIP assays using an anti-YBX1 antibody showed that YBX1 interacts with MIR155HG in A549 cells by qRT-PCR. D Immunoblot detection of the YBX1 protein in A549 cells as retrieved by in vitro transcribed biotinylated RNAs of different constructs of MIR155HG or its antisense sequence (negative control). E RIP assays were performed using an anti-Flag antibody in A549 cells transfected with different constructs of YBX1. qRT-PCR was used to measure the enrichment of MIR155HG. Western blot was used to evaluate the expression of different constructs of YBX1. F Western blot analysis of YBX1 and GAPDH in A549 and PC9 cells with MIR155HG knockdown or overexpressed. G The protein levels of YBX1 were measured in MIR155HG-overexpressing A549 cells by western blot. Cells were treated with CHX (50 mg/mL) for 3 or 6 h before harvest. H The protein levels of YBX1 were checked in sh-MIR155HG A549 cells by western blot. Cells were treated with MG132 (20 mmol/L) for 3 h before harvest. I A549 cells were transfected with Flag-YBX1, Myc-Ub, or MIR155HG plasmid and treated with MG132 (20 mmol/L) for 3 hours. The ubiquitinylated YBX1 was measured by western blot using an anti-Ub antibody following the immunoprecipitation of Flag-PKM2 with an anti-flag antibody. Results are presented as mean ± SEM, n = 3. ***p < 0.001.