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. Author manuscript; available in PMC: 2019 May 4.
Published in final edited form as: Cell Rep. 2018 May 15;23(7):2083–2094.e6. doi: 10.1016/j.celrep.2018.04.060

Figure 6. tBID Does Not Promote BOK-Mediated Cell Death.

Figure 6.

(A) Schematic of the Tet-On 3G stable expression system of Venus-2A-HA-mouse BOK in bak−/− bax−/− MEFs. We typically preincubate cells with Dox for up to 24 hr, which initiates the production of BOK mRNA, and induce cell death with MG132, which inhibits the proteasome, thereby stabilizing BOK protein.

(B) Transient transfection of vectors expressing mCherry (mC) and tBID-mCherry (tBID-mC) was performed according to the scheme in Figure S6A. After recovery in the respective doses of Dox, cell death was induced with MG132 for 18 hr. Western blotting of samples treated with the caspase inhibitor qVD was conducted sequentially with BID, GFP, BOK, and actin antibodies. The blue arrow in the BID blot indicates cross-reactivity by the BID antibody with a band that migrates slightly faster than tBID-mCherry. Weak production of tBID alone also is detected, perhaps being generated through proteolysis of tBID-mCherry. The banding pattern indicates potential BOK ubiquitination.

(C) Cell death induced by WT mBOK in (B) was monitored hourly by IncuCyte imaging of SYTOX Green staining. The 18 hr time point indicates insignificant differences between the mCherry and tBID-mCherry samples at each Dox concentration. Data represent average and SD of triplicate. See also Figure S6.