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. 2013 Mar 7;49(5):959–971. doi: 10.1016/j.molcel.2012.12.022

Figure 5.

Figure 5

Activation of FAK and Akt Leads to the Dissociation of Mitochondrial Bax in Detached Cells

(A) MECs transiently expressing myrFAKER (upper panel) or myrAktER (lower panel) were treated with 4-OHT for the indicated times. Lysates were immunoblotted with the indicated antibodies. IP, immunoprecipitation.

(B) Reattachment of MECs leads to the rapid activation of FAK and Akt. MECs were analyzed by immunoblotting for phosphorylation of FAK on tyrosine 397 and Akt on serine 473.

(C) MECs transiently expressing GFP-Bax alone or in combination with myrAktER or myrAkt (expressed from the same plasmid) were detached for 30 min. Cells were left untreated for an additional 30 min or treated with 4-OHT for 30 min, then immunostained for anti-GFP and anti-mtHsp70. The proportion of cells with predominantly mitochondrial GFP-Bax was quantified. The mean of three independent experiments is shown. Error bars represent SEM. Data were analyzed by ANOVA; ∗∗∗ = p < 0.0001. The panel on the right shows representative images.

(D) Adherent MECs coexpressing GFP-Bax and either myrAktK179M or myrAktK179MER were untreated or treated with 4-OHT. The distribution of GFP-Bax was determined as in (C). The mean of three independent experiments is shown. Error bars represent SEM. Data were analyzed by ANOVA; ∗∗ = p < 0.01. Representative images are shown on the right.

(E) MECs expressing GFP-Bax and myrAktK179MER were left untreated or treated with 4-OHT, followed by 8 hr either with or without washing out the 4-OHT. The subcellular distribution of GFP-Bax was then quantified as in (C). Error bars represent SEM.