Fig. 3: Specificity and selectivity of Compound-c2 for HIF2α.

a Caki-2 cells were treated with 30 μM Compound-c2 for the indicated durations. The stability of HIF1α was assessed by immunoblotting. β-actin was used as a loading control. LE Long Exposure, SE Short Exposure. b Lysate from 786-O cells was challenged with the indicated amounts of Biotin-Compound-c2 for 1 h. HIF2α binding and input was assessed by immunoblotting. c Empty vector (EV) and HA-HIF2α were transiently expressed in HEK293 cells and lysates were challenged with the indicated amounts of Biotin-Compound-c2 for 1 h. HIF2α binding to Compound-c2 was assessed by immunoblotting. d Amino acid sites of the HIF2α PAS-B domain predicted to contact Compound-c2. Residues shown are those within 3.5 Å of the docked model of Compound-c2. e HEK293 cells were transiently transfected with indicated HA-HIF2α mutants. Lysates were challenged with 1 µM of Biotin-Compound-c2 for 1 h and HIF2α binding to Compound-c2 was assessed by immunoblotting. Densitometry was performed using Photoshop v.23.5.1 to quantify Western blot band signal intensity. Bars represent the mean signal intensity values of HIF2α pulldown:HIF2α input compared to that of WT for three independent measurements. Error bars represent the standard deviation (S.D.) of three independent measurements. A Student’s t test (two-sided) was performed to assess statistical significance compared to WT control (NS- not significant). (See also Fig. S3B-C).