Figure 4
Figure 4. CtBP1 is not essential for the stability or the activity of the FA core complex. (A) Functional FA core complex assembly in cells depleted of CtBP. Co-immunoprecipitations were performed in HEK293T cells transduced with shRNA against CtBP1/CtBP2 (CtBPi) or with shRNA against scrambled sequences (control). Protein extracts were collected 3 days post-transductions and were subjected to IP with anti-FANCA, anti-FANCE, anti-FANCD2, and anti-CtBP1 antibodies. The IPs were resolved by SDS-PAGE and analyzed via IB with the indicated antibodies. Negative IP control was performed using rabbit IgG (R). Results shown are representative of 2 independent experiments. (B) CtBP1 is not required for FA core complex activity. HeLa cells transduced with shRNA against CtBP1 (CtBP1i), CtBP2 (CtBP2i), CtBP1/CtBP2 (CtBPi), FANCA (FANCAi) or FANCA/CtBP1/CtBP2 (CtBPi/FANCAi) were treated with 300 nM MMC or 2 mM HU for 16 hours. Equal amounts of protein were subjected to IB analysis. The FANCD2-Ub:FANCD2 ratios (L:S) are indicated below each sample. Controls included untreated cells (not shown) and cells transduced with shRNA against scrambled sequences (Control). Results shown are representative of 5 separate experiments. (C) CtBP1 is not required for FANCD2 foci formation. CtBP1- (CtBP1i) and FANCA- (FANCAi) depleted HeLa cells were treated with 150 nM MMC or 2 mM HU and subjected to immunofluorescence (IF) analysis and visualized using a Nikon E800 microscope equipped with C1 confocal system (100× magnification). White boxes in the left panels indicate selected cells magnified in the right panel for each cell line. (D) FANCD2 foci formation in CtBP-depleted cells. HeLa cells depleted of CtBP1 and/or CtBP2 (CtBP1i, CtBP2i, CtBPi), FANCA (FANCAi) or CtBP1/CtBP2 and FANCA (CtBPi/FANCAi) were treated as in panel C. Cells containing more than 10 bright foci were scored as FANCD2 foci-positive cells. Fluorescence intensity settings were determined using untreated cells. A minimum of 150 cells was scored for each condition. Values are expressed as the mean percentages of FANCD2 foci positive cells ± standard error of the mean (SEM). Ctl, control. ***P ≤ .001.

CtBP1 is not essential for the stability or the activity of the FA core complex. (A) Functional FA core complex assembly in cells depleted of CtBP. Co-immunoprecipitations were performed in HEK293T cells transduced with shRNA against CtBP1/CtBP2 (CtBPi) or with shRNA against scrambled sequences (control). Protein extracts were collected 3 days post-transductions and were subjected to IP with anti-FANCA, anti-FANCE, anti-FANCD2, and anti-CtBP1 antibodies. The IPs were resolved by SDS-PAGE and analyzed via IB with the indicated antibodies. Negative IP control was performed using rabbit IgG (R). Results shown are representative of 2 independent experiments. (B) CtBP1 is not required for FA core complex activity. HeLa cells transduced with shRNA against CtBP1 (CtBP1i), CtBP2 (CtBP2i), CtBP1/CtBP2 (CtBPi), FANCA (FANCAi) or FANCA/CtBP1/CtBP2 (CtBPi/FANCAi) were treated with 300 nM MMC or 2 mM HU for 16 hours. Equal amounts of protein were subjected to IB analysis. The FANCD2-Ub:FANCD2 ratios (L:S) are indicated below each sample. Controls included untreated cells (not shown) and cells transduced with shRNA against scrambled sequences (Control). Results shown are representative of 5 separate experiments. (C) CtBP1 is not required for FANCD2 foci formation. CtBP1- (CtBP1i) and FANCA- (FANCAi) depleted HeLa cells were treated with 150 nM MMC or 2 mM HU and subjected to immunofluorescence (IF) analysis and visualized using a Nikon E800 microscope equipped with C1 confocal system (100× magnification). White boxes in the left panels indicate selected cells magnified in the right panel for each cell line. (D) FANCD2 foci formation in CtBP-depleted cells. HeLa cells depleted of CtBP1 and/or CtBP2 (CtBP1i, CtBP2i, CtBPi), FANCA (FANCAi) or CtBP1/CtBP2 and FANCA (CtBPi/FANCAi) were treated as in panel C. Cells containing more than 10 bright foci were scored as FANCD2 foci-positive cells. Fluorescence intensity settings were determined using untreated cells. A minimum of 150 cells was scored for each condition. Values are expressed as the mean percentages of FANCD2 foci positive cells ± standard error of the mean (SEM). Ctl, control. ***P ≤ .001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal