Results: 5

1.
Figure 2

Figure 2. Depletion of BRCA1 protein decreases HDR activity. From: The effect of BRCA1 missense mutations on homology directed recombination.

HeLa-DR13-9 cells were subjected to two rounds of siRNA transfection with and without wild-type BRCA1 expression plasmid as indicated. The I-SceI expression plasmid was transfected in lanes 2–5 and siRNA and BRCA1 expression plasmids used in transfections are indicated in the grid below the histogram. On the Y-axis, the percentages of GFP-positive cells were determined by flow cytometry. Since these results are from a single experiment, the results were not normalized.

Derek J.R. Ransburgh, et al. Cancer Res. ;70(3):988-995.
2.
Figure 1

Figure 1. The Homology-Directed Recombination Assay. From: The effect of BRCA1 missense mutations on homology directed recombination.

A. The design of the recombination substrate (15) is shown. There are two inactive alleles encoding GFP in a single locus in the genome. Transfection of the plasmid expressing the I-SceI endonuclease results in a double-stranded break. If this break is repaired by HDR using the second GFP allele as the donor homologous sequence, then the upstream copy of the gene becomes functional. B. Typical results are shown for the monolayer of cells after recombination has taken place. The effects on the numbers of cells recombined is shown for the control siRNA (top panel) and for the BRCA1-specific siRNA (bottom panel).

Derek J.R. Ransburgh, et al. Cancer Res. ;70(3):988-995.
3.
Figure 5

Figure 5. Effect of BRCA1 missense variants on association with BARD1. From: The effect of BRCA1 missense mutations on homology directed recombination.

HA-epitope tagged BRCA1 expression constructs were transfected into HEK293T cells, and two days post transfection whole cell lysates were prepared and immunoaffinity purified via the HA-epitope and analyzed by SDS-PAGE followed by immunoblot. Expression plasmids were the empty vector (lane 1), wild-type BRCA1 (lanes 2 and 14) and the indicated variant BRCA1 proteins. The H41A variant in lane 12 was not used in the HDR analysis. The top immunoblot in each panel is specific for the HA-tag, and the bottom immunoblot in each panel detects the endogenous BARD1 protein associated with the HA-tagged BRCA1 fusion protein.

Derek J.R. Ransburgh, et al. Cancer Res. ;70(3):988-995.
4.
Figure 4

Figure 4. Identification of BRCA1 missense mutations that affect homologous recombination. From: The effect of BRCA1 missense mutations on homology directed recombination.

A. Results of HDR assays with BRCA1 substitution variants. Control (lane 1) or BRCA1 3’-UTR specific (lanes 2–19) siRNA were transfected into HeLa-DR13-9 cells along with the appropriate BRCA1 expression construct, as indicated. Within a single experiment, the percentage of GFP-positive cells in the control siRNA transfected cells (lane 1) were set equal to 1.0 and results for each BRCA1 substitution variant were normalized relative to that control. B. Expression of BRCA1 substitution variants in HeLa-DR13-9 cells. Cells were transfected with the control siRNA (lane 3), BRCA1 3’-UTR specific siRNA (lanes 1, 4–22), or no transfection (lane 2). Cells were processed according to the schedule of the HDR assay, including transfection of the I-SceI expressing plasmid, and lysates were analyzed for the presence of the BRCA1 protein (top panel) or the loading control Rpb1 subunit of RNA polymerase II (bottom panel). The samples in lanes 1, 9, and 18 were from the same lysate preparation.

Derek J.R. Ransburgh, et al. Cancer Res. ;70(3):988-995.
5.
Figure 3

Figure 3. The BRCA1 N- and C-termini are important in regulation of HDR activity. From: The effect of BRCA1 missense mutations on homology directed recombination.

A. The BRCA1 deletion mutants analyzed are shown next to a ruler for the position of the corresponding amino acid residues. The yellow box from aa 1–100 is for the RING domain, and the two green boxes at the carboxy-terminus are for the tandem BRCT domains. The amino acids deleted are as follows: ΔN-BRCA1, 1–302; ΔM1-BRCA1, 305–770; ΔM2-BRCA1, 775–1292; and ΔC-BRCA1, 1527–1863. The ΔM1-BRCA1 and ΔM2-BRCA1 have nuclear localization sequences added at the site of the deletions. B. HDR assays were performed as in Figure 2 with transfected control siRNA (Con; lane 1) or BRCA1-specific siRNA (Br; lanes 2–7). BRCA1 expression vectors were transfected as indicated in the grid. In each experiment, the percentage of GFP-positive cells from control siRNA transfections were set equal to one, and the fraction of GFP-positive cells were determined relative to the control siRNA. Results are from three independent experiments and expressed as the mean +/− S.E.M. C. The Western blot shows the BRCA1 content of cells transfected in parallel with the test constructs. Endogenous BRCA1 protein was depleted from cells by transfection of a 3’-UTR specific siRNA (lanes 1–5 and 7) and cells were co-transfected with vector (lanes 1 and 6) or the indicated BRCA1 expression construct (lanes 2–5 and 7). BRCA1 content of lysates was determined by Western blot specific for BRCA1 (top panel) and protein loading control was determined by an antibody specific for alpha-tubulin (bottom panel).

Derek J.R. Ransburgh, et al. Cancer Res. ;70(3):988-995.

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