(A) Immunoblots of BAP1 deficient NCI-H226 cells. Cells were reconstituted with BAP1 WT by doxycycline (1 μg/mL, 2 days). Cycloheximide (CHX, 100 μg/mL) was treated for the indicated time. Analysis was performed to detect the indicated proteins. Ub-H2A, a known substrate of BAP1, was used as a surrogate for monitoring BAP1 activity (14) and α-Actin served as a loading control.
(B) Knock-down of hippo core components decreased the viability of BAP1 deficient NCI-H226 cells. siRNAs against YAP, TAZ, or TEAD1/2/3/4 were introduced by transfection for 72 hr and cell viability was assessed using Cell Titer-Glo. Error bars represent mean ± SD (n=3). ****P < 0.0001; Student’s t test.
(C) BAP reconstitution and knockdown of YAP/TAZ or TEADs in B was confirmed by immunoblotting with indicated antibodies.