Fig. 3

DLEU1 stabilizes the DYNLL1 protein by interfering with its ubiquitination and degradation. A Representative proteins identified by RNA pull-down and mass spectrometry using biotinylated DLEU1 sense or antisense (DLEU1-AS) RNA generated in vitro. B Immunoblotting analysis of proteins retrieved from the DLEU1 pull-down assay using a DYNLL1 antibody. C Total lysates of KYSE-150-DLEU1 cells were subjected to immunoprecipitation with anti-DYNLL1 or anti-IgG antibody followed by qRT-PCR analysis to detect DLEU1 RNA. D Immunoblot detection of DYNLL1 protein retrieved from KYSE-150-DLEU1 cells by in vitro transcribed biotinylated RNAs of the indicated DLEU1 constructs. E qRT-PCR analysis of DYNLL1 expression in KYSE-150 cells with ectopic DLEU1. F Immunoblotting analysis of the DYNLL1 protein in KYSE-150 cells overexpressing DLEU1. β-actin was used as a loading control. G DLEU1 depletion decreased the protein levels of DYNLL1. H Cells transfected with the indicated siRNAs or shRNAs were treated with CHX (50 mg/mL) for 4 or 8 h before harvest. DYNLL1 protein levels were measured by immunoblotting analysis. β-actin served as a loading control. I Cells were cultured in the presence or absence of MG132 (20 μM) after DLEU1 knockdown. DYNLL1 protein levels were checked by immunoblotting. J Lysates from KYSE-150 cells with DLEU1 overexpression and MG132 treatment were subjected to immunoprecipitation with anti-DYNLL1 antibody followed by immunoblotting with ubiquitin antibody. K KYSE-150 cells were transfected with full-length or truncated mutants of DLEU1 and were subjected to immunoprecipitation followed by immunoblotting analysis with the indicated antibodies