Figure 5 from Hypoxia-Induced Long Noncoding RNA HIF1A-AS2 Regulates Stability of MHC Class I Protein in Head and Neck Cancer

HIF1A-AS2 interacts with NBR1 and MHC-I to mediate autophagic degradation of MHC-I. A, Co-immunoprecipitation (Co-IP) to detect ubiquitination of HLA-A with or without the existence of HIF1A-AS2. 293T cells were co-transfected with HA-ubiquitin, MYC-HLA-A, and HIF1A-AS2 (pCDH-GFP-HIF1A-AS2)/or control (pCDH-GFP) for 48 hours, and bafilomycin A1 treated for 12 hours before conducting the Co-IP experiment. B, Biotinylated HIF1A-AS2 RNA pull-down assay. Biotin-labeled HIF1A-AS2 and antisense HIF1A-AS2 RNAs were incubated with the cell lysate from IFNγ treated SAS cells and pulled down by streptavidin beads. The RNA interaction proteins, NBR1 and HLA-ABC, were analyzed by western blot. C, NBR1 and HLA-ABC interaction domain on HIF1A-AS2. RNAs corresponding to indicated biotin-labeled HIF1A-AS2 were incubated with the cell lysate from IFNγ treated SAS cells, followed by streptavidin pull-down. The RNA interaction proteins, NBR1, and HLA-ABC were analyzed via western blot. D, Co-IP assay to demonstrate the interaction between HA-NBR1 and MYC-HLA-A with the existence of HIF1A-AS2. 293T cells were co-transfected with HA-NBR1, MYC-HLA-A, and HIF1A-AS2 (pCDH-GFP-HIF1A-AS2)/or control (pCDH-GFP) for 48 hours, and bafilomycin A1 treated for 12 hours before conducting the Co-IP experiment.