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Figure 1 | Cell Communication and Signaling

Figure 1

From: Chk1 activity is required for BAK multimerization in association with PUMA during mitochondrial apoptosis

Figure 1

Effect of Chk1 inhibitor on BAK activation following DNA damage. (A) BAK N-terminal conformational change was analysed in HCT116-WT BAK cells by FACS using AB1 epitope that specifically recognizes the N-terminal open conformation of BAK. BAK underwent N-terminal conformational change causes increase in BAK AB-1 specific fluorescence after UV damage that was not inhibited by prior treatment with Chk1 inhibitor SB-218078 (Calbiochem, n = 3, ± s.e.m.). (B & C) BAK multimerization assay was analysed in HCT116 DKO cells reconstituted with WT BAK as described in [6]. Mitochondria were isolated from cells treated with or without Chk1 inhibitor (SB −218078) for 30 min either alone or UV (B) radiation or etoposide treatment (C) for 8 hrs. 70–100 μg of mitochondria were crosslinked with 10 mM BMH (top panel) or 10 mM BMOE (bottom panel). BAK was detected by rabbit anti BAK monoclonal ab (abcam Y164). The input was the 5% of mitochondrial extracts used in the crosslinking studies to ensure equal loading (middle panel). Non cross-linked BAK runs as a monomer (M) and also as an intra-molecularly linked monomer (Mx). BAK dimers (D), trimers (T) and higher order structures are indicated. Note that BMOE only detects monomer and dimer forms of BAK. This is representative of 3 independent experiments. Similarly, multimerization experimemts were performed with BAK Y108A mutant (D), BAK S117A mutant (E) and BAK S117E (F) mutant in the presence of the Chk1 inhibitor and ± UV.

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