Journal: Oncogene

Article Title: Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

doi: 10.1038/sj.onc.1207895

Figure Lengend Snippet: Figure 4 Depletion of hSNM1B by siRNA does not affect monoubiquitination of FANCD2. SV40-transformed fibroblasts (GM0637) were transfected with hSNM1B, FANCA or a negative control siRNA and assayed 66 h later for monoubiquitination of FANCD2 by immunoblot. Cells were either untreated or treated with MMC or IR as indicated

Article Snippet: Polyclonal Nibrin/p95 (NB100-143) and FANCD2 (NB 100- 182) antibodies were purchased from Novus-Biologicals (Littleton, CO, USA).

Techniques: Transformation Assay, Transfection, Negative Control, Western Blot

Journal: Oncogene

Article Title: Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

doi: 10.1038/sj.onc.1207895

Figure Lengend Snippet: Figure 3 Depletion of hSNM1B from HeLa cells increases sensitivity to ICL-inducing agents and to IR. (a) Clonogenic survival of HeLa cells transfected with hSNM1B siRNA, FANCD2 siRNA or control siRNA after treatment with increasing concentrations of MMC. (b) Survival of hSNM1B-depleted and control siRNA-transfected cells after treatment with cisplatin or (c) survival of HeLa cells transfected with hSNM1B siRNA, 53BP1 siRNA or control siRNA after treatment with increasing doses of IR. A fraction of HeLa cells treated with siRNAs for use in survival experiments was assayed for protein expression. Whole-cell lysates were immunoblotted for FANCD2 (d) or for 53BP1 (e). p95/nibrin expression was assayed as a control for specificity of RNA interference

Article Snippet: Polyclonal Nibrin/p95 (NB100-143) and FANCD2 (NB 100- 182) antibodies were purchased from Novus-Biologicals (Littleton, CO, USA).

Techniques: Transfection, Control, Expressing

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: FANCD2 protein expression in MG-63 cells after RNAi. Lane 1: Control; Lane 2: siRNA-Control; Lane 3: siRNA-FANCD2 24 h; Lane 4: siRNA-FANCD2 48 h.

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Expressing, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Absorbance of MG-63 cells after siRNA-FANCD2 interfere by CCK-8 assay ( x ̅ ±s, n = 6)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: CCK-8 Assay, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Cell cycle distribution of MG-63 cells after siRNA-FANCD2 interfere ( x ̅ ±s, n = 4)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Apoptotic percentages of MG-63 cells after FANCD2 siRNA interfere by Flow Cytometry ( x ̅ ±s, n = 4)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Flow Cytometry, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Relative expression of mRNAs in MG-63 cells after FANCD2 siRNA interfere ( x ̅ ±s, n = 3)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Expressing, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Western blotting picture of p53, phos-p53, p21, TP53INP1, cleaved caspase-9 and-3 protein expression after RNAi. Lane 1: Control; Lane 2: siRNA-Control; Lane 3: siRNA-FANCD2 24 h; Lane 4: siRNA-FANCD2 48 h.

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Western Blot, Expressing, Control

Journal: Oncotarget

Article Title: FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation

doi: 10.18632/oncotarget.9595

Figure Lengend Snippet: A. , B. Overexpression of GSK3β and FBW7 suppresses damage-induced FANCD2 monoubiquitination and foci formation. A. HeLa cells coexpressing HA-tagged GSK3β and FBW7 were treated with 1 μM MMC for 8 h and cell lysates analyzed by Western blotting. B. U2OS cells coexpressing HA-tagged GSK3β and FBW7 were treated with 100 ng/mL MMC for 16 h and subjected to anti-FANCD2 immunofluorescence. C. Quantification of cells in B. exhibiting more than 10 FANCD2 foci. Data shown are the mean ± SD from three independent experiments. * p < 0.01 compared with vector control. D. GSK3β and FBW7 overexpression facilitates the turnover of FANCA and FANCG. HeLa cells expressing HA-tagged GSK3β and FBW7 were treated with 50 μg/mL CHX for the indicated times and analyzed by Western blotting. E. Densitometry of FANCA and FANCG levels in D. quantitated by ImageJ. F. GSK3β and FBW7 overexpression sensitizes cells to a DNA interstrand cross-linking agent. U2OS cells expressing HA-GSK3β and HA-FBW7 were plated to 96 wells, treated with the indicated doses of MMC for 5 days, and cell viability was measured by luminescence assay. Data shown are the mean ± SD from three independent experiments. * p < 0.05 compared with control.

Article Snippet: Cells were incubated with an anti-FANCD2 primary antibody (1: 500, Novus Biologicals) in PBS/ 1 % BSA for 2 h at RT, washede three times in PBS, and incubated with 1:1000 Alexa Fluor ® 568 goat anti-mouse IgG secondary antibody (Molecular Probes) for 1 h at RT.

Techniques: Over Expression, Western Blot, Immunofluorescence, Plasmid Preparation, Control, Expressing, Luminescence Assay

Journal: Oncotarget

Article Title: FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation

doi: 10.18632/oncotarget.9595

Figure Lengend Snippet: A. Depletion of FBW7 hypersensitizes cells to a DNA interstrand cross-linking agent. U2OS cells transfected with indicated siRNA for 48 h were plated to 96 wells, treated with the indicated doses of MMC for 5 days, and cell viability was measured by luminescence assay. FAAP20 depletion served as a positive control. Data shown are the mean ± SD from three independent experiments. * p < 0.05 compared with control. B. A schematic for the FAAP20 knockout strategy using CRISPR/Cas9. The 20-nucleotide sgRNA target loci in the exon 1 are marked in blue line along with a PAM sequence in red. The cleavage site for the Cas9 nuclease is shown by red triangle. The ATG start codon is marked in bold with arrow. C. U2OS wild-type (vector transfected) or FAAP20 knockout (KO) clones were treated with 100 ng/mL MMC for 16 h and analyzed by Western blotting. D. Western blot analyses of U2OS FAAP20 KO cells reconstituted with FAAP20 wild-type or SA mutant by retroviral transduction. E. Restoration of FANCD2 monoubiquitination by exogenous FAAP20 wild-type or SA mutant. FAAP20 KO cells stably expressing FAAP20 wild-type or SA mutant were treated with 100 ng/mL MMC for 16 h and analyzed by Western blotting. F. Accumulation of FANCA and FANCD2 monoubiquitin in the chromatin-enriched fraction in cells expressing the FAAP20 SA mutant. Indicated U2OS cells were treated with 1 μM MMC for 2 h, replenished with fresh medium to initiate the DNA repair process, and collected at the indicated times. Cells were fractionated, and chromatin-enriched fractions were analyzed by Western blotting. Asterisks denote nonspecific bands. G. The half-life of FANCA in the chromatin extends in the cells expressing the FAAP20 SA mutant. (Top) U2OS FAAP20 KO cells expressing FAAP20 wild-type or SA mutant were treated with 100 ng/mL MMC for 16 h, incubated with 50 μg/mL CHX for the indicated times and fractionated to isolate chromatin-enriched fractions. Cell lysates were analyzed by Western blotting. (Bottom) Quantification of the FANCA level normalized by ORC2. Error bars indicate SD from two independent experiments. * p < 0.05 compared with SA. H. U2OS cells serially transfected with siRNA and siRNA-resistant FAAP20 variants (siR*) were treated with indicated doses of MMC, and cell viability was measured by luminescence assay. Data shown are the mean ± SD from three independent experiments. * p < 0.05 (WT and SA) compared with control except 125 nM for SA ( p = 0.4940 not significant).

Article Snippet: Cells were incubated with an anti-FANCD2 primary antibody (1: 500, Novus Biologicals) in PBS/ 1 % BSA for 2 h at RT, washede three times in PBS, and incubated with 1:1000 Alexa Fluor ® 568 goat anti-mouse IgG secondary antibody (Molecular Probes) for 1 h at RT.

Techniques: Transfection, Luminescence Assay, Positive Control, Control, Knock-Out, CRISPR, Sequencing, Plasmid Preparation, Clone Assay, Western Blot, Mutagenesis, Retroviral, Transduction, Stable Transfection, Expressing, Incubation

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet: Identification of a Phosphorylation Cluster on FANCD2 (A) Schematic representation of the generation of a Flag-HA-FANCD2 knock-in HeLa cell line through the use of CRISPR/Cas9. Exons are shown in green, and Flag-HA tag is shown in red. (B) Flag purification of both FANCD2 and Ub-FANCD2 from the knock-in HeLa cell line. Immunoblot analysis showing whole-cell lysate (WCL), pellet, lysate, flowthrough (FT), and elution of the purification. Silver stain showing the four elution products used for MS/MS in unperturbed conditions (no TMP) and after the induction of ICLs with TMP (TMP). (C) Alignment of residues 874–905 of human FANCD2 protein to those in mouse ( Mus ), chicken ( Gallus ), toad ( Xenopus ), zebrafish ( Danio ), and fruit fly ( Drosophila ). Serine and threonine residues are in red, and aspartic and glutamic acid residues are in blue (alignments done with ClustalW2). (D) Relative intensity of ubiquitinated peptides to unmodified peptides as identified in MS/MS in four samples: no ubiquitination/no TMP (1), ubiquitination/no TMP (2), no ubiquitination/TMP (3), and ubiquitination/TMP (4). Mean ± SD in n = 2 independent experiments. (E) Relative intensity of phosphorylated peptides to unmodified peptides (containing residues 882, 884, 886, 891, 896, and 898) as identified in MS/MS in four samples: no ubiquitination/no TMP (1), ubiquitination/no TMP (2), no ubiquitination/TMP (3), and ubiquitination/TMP (4). Mean ± SD in n = 2 independent experiments. (F) Crystal structure of the mouse FANCD2/FANCI complex (marked in yellow; PDB: 3S4W ) docked into the cryo-EM structure of the human FANCD2/FANCI complex (in gray; EMDB: EMD-8141) showing the approximate location of the 882–898 cluster (marked in red) as well as the C-terminal Tower domain of FANCD2, where some DNA binding residues have been found. It should be noted that residues 882–898 were deleted from the mouse FANCD2 protein used to obtain the crystal structure.

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: Knock-In, CRISPR, Purification, Western Blot, Silver Staining, Tandem Mass Spectroscopy, Cryo-EM Sample Prep, Binding Assay

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet: Phosphorylation of the 882–898 Cluster on FANCD2 Suppresses Activation of the FANCD2/FANCI Complex in Human Cells (A) Survival assay to the crosslinking agent mitomycin C (MMC) added at the indicated concentrations and left for 2 weeks. Survival is assessed as the number of colonies formed after 2 weeks. HeLa cells were used, FANCD2 was knocked out with CRISPR/Cas9, and the HeLa FANCD2−/− cells were stably complemented with EGFP-FANCD2, EGFP-FANCD2-6A, or EGFP-FANCD2-6D (mean ± SEM, n = 3). (B) Immunoblot analysis of cell lysates of the HeLa FANCD2−/− cells complemented with EGFP-FANCD2, EGFP-FANCD2-6A, or EGFP-FANCD2-6D before and after treatment with TMP (2 μg/mL) and UVA (50 mJ/cm 2 ) for 3 h. (C) Immunoblot analysis of cell lysates of HeLa FANCD2−/− cells complemented with EGFP-FANCD2, EGFP-FANCD2-6A, or EGFP-FANCD2-6D before and after treatment with TMP (2 μg/mL) and UVA (50 mJ/cm 2 ) for 3 h. Samples were fractionated with CSK buffer so chromatin-bound and soluble fractions could be separated. (D) Live cell imaging of HeLa FANCD2−/− cells complemented with EGFP-FANCD2, EGFP-FANCD2-6A, or EGFP-FANCD2-6D and mCherry-UHRF1. Cells were treated with TMP (20 μg/mL) and microirradiated at the indicated areas (white arrows) and followed for the indicated times (stripe intensity quantified as mean ± SEM, n = 5) (scale bar, 10 μm).

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: Activation Assay, Clonogenic Cell Survival Assay, CRISPR, Stable Transfection, Western Blot, Live Cell Imaging

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet: CK2 Phosphorylates FANCD2 in the 882–898 Cluster In Vivo and Reduces Its Monoubiquitination In Vitro (A) Flag purification of endogenous FANCD2 from HeLa S3 knock-in cell line. Ctr, untreated control; CX-4945, treated with 10 μM CK2 inhibitor CX-4945 for 18 h. (B) Relative intensity of phosphorylated peptides containing the 882–898 cluster on FANCD2 in either untreated control or treated with the CK2 inhibitor CX-4945 for 18 or 48 h. (C) Coomassie blue gel of the proteins used in the in vitro ubiquitination assay: Flag-HA-UBA1 (E1), UBE2T (E2), Flag-HA-FANCL (E3), and His-ubiquitin. (D) Coomassie blue gel of the purification of the Flag-HA-FANCD2/His-FANCI complex co-expressed in Sf9 cells. (E) Coomassie blue gel of the in vitro ubiquitination of the FANCD2/FANCI complex as WT, 6A, or 6D forms. Quantification showing the ratio of Ub-FANCD2 to FANCD2. (F) Coomassie blue gel of the in vitro ubiquitination of the WT, 6A, and 6D forms of the FANCD2/FANCI complex following a mock treatment or in vitro phosphorylation by CK2. Quantification showing the ratio of Ub-FANCD2 to FANCD2.

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: In Vivo, In Vitro, Purification, Knock-In, Ubiquitin Assay

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet: Phosphorylation of the 882–898 Cluster on FANCD2 Hinders Its DNA Binding and Can Be Reversed by Dephosphorylation (A) Electrophoretic mobility shift assay (EMSA) showing the DNA binding of the Flag-HA-FANCD2/His-FANCI complex (WT, 6A, or 6D form) to a Y-shaped radiolabeled DNA probe (representative experiment of n = 2). Quantification showing intensities of the protein/DNA complexes. (B) EMSA showing the DNA binding of the Flag-HA-FANCD2/His-FANCI complex (WT and 6A) after mock or CK2 treatment to a Y-shaped radiolabeled DNA probe (loading control in Figure S4 A) (representative experiment of n = 2). Quantification showing intensities of the protein/DNA complexes. (C) EMSA showing the DNA binding of the Flag-HA-FANCD2/His-FANCI complex (WT) after mock, CK2, λPP or both treatments to a Y-shaped radiolabeled DNA probe (representative experiment of n = 2). Quantification showing intensities of the protein/DNA complexes.

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: Binding Assay, De-Phosphorylation Assay, Electrophoretic Mobility Shift Assay

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet: Lack of Phosphorylation of the 882–898 Cluster on FANCD2 Leads to Enhanced Recruitment to ICLs and Ubiquitination In Vivo (A) Cell cycle profiles measured by DNA content of HeLa FANCD2−/− cells and HeLa FANCD2−/− cells complemented with FANCD2-WT, FANCD2-6A, and FANCD2-6D after no treatment (Ctr) or after treating with 20 ng/mL MMC for 2 h and recovering for 24 h (MMC). Graph shows the percentage of cells in G2 for each cell line and treatment. (B) Live-cell imaging of HeLa FANCD2−/− cells complemented with EGFP-FANCD2 or EGFP-FANCD2-6A and mCherry-UHRF1. Cells were treated with TMP (20 μg/mL) and microirradiated at the indicated areas (white arrows) and followed for the indicated times (stripe intensity quantified as mean ± SEM, n = 5) (scale bar, 10 μm). (C) Immunoblot analysis of cell lysates of HeLa FANCD2−/− cells complemented with Flag-HA-FANCD2 or Flag-HA-FANCD2-6A either asynchronous (AS) or synchronized with double thymidine block (S and G1). ICLs were introduced with TMP (2 μg/mL) and UVA (50 mJ/cm 2 ) 1.5 h before harvest.

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: In Vivo, Live Cell Imaging, Western Blot, Blocking Assay

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet: Model of the Regulation of the FA Pathway by CK2 Phosphorylation of the FANCD2/FANCI Complex CK2 phosphorylates FANCD2 constitutively on the 882–898 cluster and prevents DNA binding of the FANCD2/FANCI complex in the absence of DNA damage. Upon the appearance of DNA damage, FANCD2 is dephosphorylated, increasing the affinity of the FANCD2/FANCI complex to DNA. This form of the FANCD2/FANCI complex can be considered facultative active. Once bound to DNA, the FANCD2/FANCI complex is monoubiquitinated by the core complex containing the E3 ligase FANCL, bringing the complex to its fully active state. Monoubiquitination locks the FANCD2/FANCI complex on DNA, completing its activation, and allows ICL repair to initiate.

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: Binding Assay, Activation Assay

Journal: Cell Reports

Article Title: Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

doi: 10.1016/j.celrep.2019.05.003

Figure Lengend Snippet:

Article Snippet: Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM ATP and 2 mM DTT.

Techniques: Recombinant, Knock-In, CRISPR

Journal: Alcoholism, clinical and experimental research

Article Title: Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia-breast cancer susceptibility (FA-BRCA) DNA damage response network.

doi: 10.1111/j.1530-0277.2011.01563.x

Figure Lengend Snippet: Fig. 3. Ethanol exposure of HeLa-ADH1B cells stimulates FANCD2 monoubiquitination. (A) Cells were exposed to 20 mM ethanol or 2 mM hydroxyurea (HU) and whole cell extracts probed with anti-FANCD2. The positions of the long (L) and short (S) forms of FANCD2 are indicated. The ratio of the L and S forms of FANCD2 was determined using the NIH ImageJ program and results shown at the right. (B) Cells were exposed to media alone, 20 mM ethanol, or 20 mM ethanol in the presence of different concentrations of 4-methyl pyrazole (4-MP), and extracts were analyzed for FANCD2 monoubiquitination. Quantification is shown at the right.

Article Snippet: Blots were probed with antibodies against FANCD2 (Novus Biologicals, Littleton, CO), BRCA1, or BRCA1 pSer 1524 (both from Bethyl Laboratories, Montgomery, TX).

Techniques:

Journal: Alcoholism, clinical and experimental research

Article Title: Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia-breast cancer susceptibility (FA-BRCA) DNA damage response network.

doi: 10.1111/j.1530-0277.2011.01563.x

Figure Lengend Snippet: Fig. 5. FANCD2 monoubiquitination and BRCA1 phosphorylation following ethanol exposure in HeLa-ADH1B and HeLa-ADH1B-ALDH2 cells. Increased FANCD2 monoubiquitination is seen in both HeLa-ADH1B cells and HeLa-ADH1B-ALDH2 cells after ethanol exposure, whereas ethanol increased BRCA1 phosphorylation at Ser 1524 only in HeLa-ADH1B cells.

Article Snippet: Blots were probed with antibodies against FANCD2 (Novus Biologicals, Littleton, CO), BRCA1, or BRCA1 pSer 1524 (both from Bethyl Laboratories, Montgomery, TX).

Techniques: Phospho-proteomics