BRCA1 Forms a Functional Complex with -H2AX as a Late Response to Genotoxic Stress
Figure 4
BRCA1 knockdown stabilizes -H2AX and reduces ubiquitination of -H2AX. (a) Cells treated with different amounts of antisense morpholino oligos (AS1 and AS2) or a scrambled antisense morpholino (scrAS) were stained with DAPI or immunostained for -H2AX. A second set of morpholino-treated cells was immunostained for BRCA1 to show dose-dependent knockdown of BRCA1 protein. (b) Cells treated with scrambled antisense morpholino (lane 1, scrAS) or increasing amounts of antisense morpholino oligos (AS1/2, lanes 2 and 3) were lysed and separated by SDS-PAGE and probed (WB) for BRCA1, -H2AX, -actin, or -tubulin. (c) 293T cells were treated with control (scrambled antisense (scrAS) or BRCA1 antisense morpholino oligos (AS1/2) and then transfected with control vector, HA-ubiquitin alone, and/or H2AX-E139-V5-H6. Histidine- (H6-) tagged H2AX was purified from chromatin fractions then probed for HA-ubiquitin. The blot was stripped and reprobed for V5-tag on H2AX. (d) Cells treated with a scrambled antisense morpholino (scrAS) or anti-BRCA1 morpholino oligos (AS1/2) were stained with DAPI (i–iv) or immunostained for -H2AX (i’–iv’). Micrographs were captured with either 10x (i–iii) or 60x (iv) objectives. Final magnification was 100x or 600x.
(a) BRCA1 loss stabilizes -H2AX
(b)
(c) BRCA1 knockdown reduces ubiquitination of -H2AX mimetics
