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. 2021 Mar 25;17(5):1263–1276. doi: 10.7150/ijbs.56357

Figure 5.

Figure 5

HLA-F was involved in mediating HK2 protein stabilization. (A) C8-D1A cells were infected with either empty lentivirus or HLA-F lentivirus and selected with puromycin for 14 days. The relative expression of Hk2 in C8-D1A cells infected with either empty lentivirus or HLA-F lentivirus was examined by real-time RT-PCR. n.s., not significant. (B) Actinomycin D was added into the wells for the indicated timepoints to block RNA synthesis. Hk2 mRNA was analyzed using real-time RT-PCR in C8-D1A cells infected with either empty lentivirus or HLA-F lentivirus. (C) The relative expression of Hk2 in HS683 cells transfected with either control shRNA or shRNA against HLA-F was examined by real-time RT-PCR. n.s., not significant. (D) Actinomycin D was added for the indicated timepoints. Hk2 mRNA was analyzed using real-time RT-PCR in HS683 cells transfected with either control shRNA or shRNA against HLA-F. (A-D) The data represent one of three independent experiments performed in triplicate with similar results. Significance was calculated using Student's t-test. (E) C8-D1A cells infected with either empty lentivirus or HLA-F lentivirus were treated with CHX at a final concentration of 100 μg/mL. At the indicated timepoints, protein was extracted, and the levels of flag, HK2, and β-actin were analyzed by Western blotting. The amount of HK2 in these cells was quantified by densitometry, normalized to the level of β-actin, and plotted. The data shown are representative of one of two independent experiments. (F) HS683 cells transfected with either shCtrl or shHLA-F were treated with CHX for the indicated timepoints. Protein was extracted, and the levels of HLA-F, HK2 and β-actin were analyzed by Western blotting. The amount of HK2 in these cells was quantified by densitometry, normalized to the level of β-actin, and plotted. The data shown are representative of one of two independent experiments. (G) Empty vector-expressing or HLA-F-expressing C8-D1A cells were treated with 5 μM MG-132 for 5 h before harvesting. The membrane was probed with the indicated antibodies. The data shown are representative of one of two independent experiments. (H) 293T cells were cotransfected with the indicated plasmid DNA. After 24 h, the cell lysates were isolated and immunoprecipitated with an anti-Myc antibody and analyzed by Western blotting using an anti-flag antibody.