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. 2023 Jan 19;120(4):e2217840120. doi: 10.1073/pnas.2217840120

Fig. 5.

Fig. 5.

BAP1 Deubiquitylates HIF-1α. (A) Reduced endogenous ubiquitylation of HIF-1α in HEK-293 cells co-transfected with Flag-tagged HIF-1α and Myc-tagged BAP1, catalytic inactive (C91S), or mock. Cells were treated with 10 µM MG-132 for 3 h, then total cell homogenates were collected and HIF-1α immunoprecipitated using anti-Flag resin. Ubiquitylation levels of the immunocomplexes were detected using an anti-Ub-HRP antibody and normalized on the total amount of Flag-HIF-1α immunoprecipitated (decimals indicate the ratio as per densitometric analysis). (B) Western blot analysis of in vitro ubiquitylation/de-ubiquitylation assay. HA-HIF-1α ubiquitylated in vitro, and subsequently incubated with immunopurified Myc-BAP1, Myc-BAP1(C91S), Myc-BAP1(L), Myc-BAP1(W), or mock, for 1 h. Ubiquitylation levels were detected using an anti-Ub-HRP antibody and normalized on the total amount of HA-HIF-1α (decimals indicate the ratio as per densitometric analysis).