Fig. 3: USP9X specifically interacts with HIF-2α in vivo and in vitro. | Cell Death & Disease

Fig. 3: USP9X specifically interacts with HIF-2α in vivo and in vitro.

From: USP9X integrates TGF-β and hypoxia signalings to promote ovarian cancer chemoresistance via HIF-2α-maintained stemness

Fig. 3

A Affinity purification and mass spectrometry (AP-MS) was conducted on HEK293T cells over-expressing Flag-tagged-USP9X (left) and HIF-2α (right) to analyze USP9X and HIF-2α-associated protein complexes. B, C Exogenous Co-IP were performed to examine the interaction between over-expressed Flag-USP9X and HA-HIF-2α in HEK293T cells. D, E Co-IP were performed to validate endogenous binding of USP9X and HIF-2α in CAOV3 and ID8 cells. F PLA was performed to validate colocalization of USP9X and HIF-2α in CAOV3 and ID8 cells treated with or without TGF-β1 (5 ng/mL) for 24 h. Scale bars, 20 µm. The right panels were the quantization chart. G Co-IP were performed to check interaction between different HA-tagged-HIF-2α truncation mutants (PAS-A/B, TAD-N and TAD-C) and Flag-tagged-USP9X. H Co-IP were performed to check interaction between different Flag-tagged-USP9X truncation mutants (N, U, C and D) and HA-tagged-HIF-2α. I GST pull-down assay was performed to check interaction of purified GST-tagged domain of USP9X (N, C and D) and His-HIF-2α in vitro. Data are shown as the mean ± s.d (F). P values were calculated by unpaired two-tailed Student’s t test (F). n = 3 biological independent samples (B–I).

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