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1.
Figure 3

Figure 3. From: ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

Phospho-FANCA S1449 is increased on chromatin after DNA damage and FANCA S1449A binds FANCG. (A) Chromatin extracts were prepared from HeLa cells treated with 1 μM MMC for 18 hours. Extracts were separated by SDS-PAGE and immunoblotted for phospho-FANCA S1449, total FANCA, and topoisomerase II as a loading control. (B) Whole cell extracts from HeLa cells expressing Flag-FANCA, Flag-FANCA S1449A, or the vector control and treated with 1 μM MMC for 6 hours were immunoprecipitated with anti-Flag. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted for FANCA and FANCG. topoII indicates topoisomerase II; P-S1449, phosphoserine 1449; and IP, immunoprecipitation.

Natalie B. Collins, et al. Blood. 2009 March 5;113(10):2181-2190.
2.
Figure 5

Figure 5. From: ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

FANCA serine 1449 phosphorylation is dependent on ATR in vivo. (A) GM6914 cells expressing wild-type Flag-FANCA, Flag-FANCA (S1449A), or the vector control were treated with 0.1 μM MMC for 20 hours. At 16 hours, 1 μM wortmannin was added (lanes 2, 4, and 6) for 4 hours. Whole cell lysates were separated by SDS-PAGE and immunoblotted for phospho-FANCA S1449 and total FANCA. (B) Wild-type (GM02188), ATR-Seckel (DK0064), and ATM-deficient (GM01389D) cell lines were treated with 50 nM MMC and the indicated dose of wortmannin for 18 hours before preparation of lysates. Western blotting was performed with the phosphospecific or nonspecific antibodies to FANCA. P-S1449 indicates phosphoserine 1449.

Natalie B. Collins, et al. Blood. 2009 March 5;113(10):2181-2190.
3.
Figure 2

Figure 2. From: ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

FANCA is induced after MMC treatment but is not phosphorylated during S phase. (A) Exponentially growing HeLa cells were treated with 1 μM MMC for the time indicated, before preparation of whole cell lysates and SDS-PAGE electrophoresis. Western blotting was performed with the phospho-specific or nonspecific antibodies to FANCA as indicated. (B) Conditions were as in panel A, with MMC treatment continued for the indicated time. (C). HeLa cells were synchronized by double thymidine block at the G1/S border (lane 4) and released for 3 hours into S phase (lane 5) or 6 hours into late S/G2 phase (lane 6). Alternatively, cells in lane 2 were treated with 1 μM MMC for 18 hours. Whole cell extracts were separated by SDS-PAGE and immunoblotted for phosphoserine 1449, FANCA, FANCD2, and Ku86 as a loading control. P-FANCA indicates phospho-FANCA.

Natalie B. Collins, et al. Blood. 2009 March 5;113(10):2181-2190.
4.
Figure 1

Figure 1. From: ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

Phospho-FANCA serine 1449 is induced after DNA damage. (A) HeLa cells treated with 1 μM MMC for 18 hours (lanes 2-5) and 1 μM okadaic acid for 30 minutes (lane 3) were lysed, and 175 μg protein was subjected to SDS-PAGE and immunoblot for endogenous FANCA. In lanes 4 and 5, 275 μg protein from HeLa cells treated with MMC was treated directly with λ-phosphatase and run on SDS-PAGE. Proteins were immunoblotted using antiphosphoserine 1449 antibody. FANCA immunoblot shows even FANCA expression. β-tubulin shows equal loading. All lanes depicted are from the same gel, same exposure. (B) HeLa cells expressing Flag-tagged FANCA, Flag-FANCA (S1449A), or vector control were treated with MMC 1 μM for 18 hours, lysed, run on SDS-PAGE, and immunoblotted using the same antibodies as in panel A. (C) Cell lysates from cells in panel B were immunoprecipitated with anti-Flag antibody and subjected to SDS-PAGE and immunoblot using antiphospho-(Ser/Thr) ATM/ATR substrate antibody. IP indicates immunoprecipitation; Ppase, phosphatase; P-S1449, phosphoserine 1449; P-L(S/T)Q, phospho-leucine (serine/threonine) glutamine (ATM/ATR substrate); and MW, molecular weight marker.

Natalie B. Collins, et al. Blood. 2009 March 5;113(10):2181-2190.
5.
Figure 4

Figure 4. From: ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

FANCA S1449A fails to completely correct FA-associated phenotypes. (A) Results of 3 growth inhibition assays are shown for GM6914 (FA-A) cells expressing wild-type Flag-FANCA, Flag-FANCA (S1449A), or the vector control. (B) Nuclear extracts were prepared from the same cells as in panel A treated with 0.1 μM MMC for 18 hours. Extracts were separated by SDS-PAGE and immunoblotted for FANCD2. Ku86 serves as a loading control. L/S ratios were calculated using densitometric measurements of short and long band intensities. (Bottom panel) The same cells as in panel A were treated with 0.1 μM MMC for the indicated time and then lysed directly in SDS loading buffer, separated by SDS-PAGE, and immunoblotted for FANCD2 and Ku86 as a loading control. L/S ratios were calculated by densitometry and plotted against time of treatment. (C) Histogram plots are shown of chromosomal aberrations seen on metaphase spreads from GM6914 cells as in panels A and B. (D) The frequency of homologous recombination in GM6914 DR-GFP cells expressing wild-type Flag-FANCA, Flag-FANCA S1449A, or the vector control was measured using flow cytometry after HDR-mediated repair of a GFP reporter substrate. Frequency is expressed as a percentage of the level of recombination seen in wild-type FANCA-expressing cells. FANCD2-L indicates long (mono-ubiquitylated) form of FANCD2; FANCD2-S, short (nonubiquitylated) form of FANCD2; and L/S Ratio, long/short ratio.

Natalie B. Collins, et al. Blood. 2009 March 5;113(10):2181-2190.
6.
Figure 6

Figure 6. From: ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

ATR phosphorylates serine 1449 of FANCA in vitro. ATR kinase was purified from HeLa cells by immunoprecipitation using anti-ATR antibody. After immunoprecipitation, beads were divided for Western blot (B) and in vitro kinase assay (A). (A) Immunoprecipitated ATR (lanes 1-3) or a control IgG immunoprecipitation (lanes 4-6) was incubated with GST (lanes 1,4), GST-FANCA C terminus (lanes 2,5), or GST-FANCA S1449A C terminus (lanes 3,6) in the presence of [γ-32P]ATP. Products were separated by SDS-PAGE, stained with Coomassie, and dried, and resulting labeled substrates were detected by autoradiography. (B) Western blot for ATR showing immunoprecipitation of ATR with anti-ATR antibody (lane 3) but not with a control IgG immunoprecipitation (lane 2). (C) Chk1 inhibition does not inhibit phospho-FANCA S1449. HeLa cells were treated for 18 hours with 1 mM MMC (lanes 2-4). Cells in lanes 3 and 4 were additionally treated with 1 μM SB218078 or 100 μM wortmannin, respectively. Whole cell lysates were separated by SDS-PAGE and immunoblotted for phospho-FANCA S1449, total FANCA, and β-actin as a loading control. (D) In vitro kinase reaction was run as in panel A, except aliquots were incubated in the presence of wortmannin. Wortmannin inhibited ATR phosphorylation of the wild-type fusion protein. C term indicates C terminus; and IP, immunoprecipitation.

Natalie B. Collins, et al. Blood. 2009 March 5;113(10):2181-2190.

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