Journal: Chromosoma

Article Title: COORDINATION OF THE RECRUITMENT OF THE FANCD2 AND PALB2 FANCONI ANEMIA PROTEINS BY A UBIQUITIN SIGNALING NETWORK

doi: 10.1007/s00412-016-0602-9

Figure Lengend Snippet: a Representative immunoblot showing knockdown of MDC1. b–c Representative images (b) and quantification (c) of FANCD2 focus formation in HeLa cells transfected with a control siRNA or two distinct siRNAs targeting MDC1 and treated with 0.5 µM MMC for 16 hr. FANCD2 foci (red) are also shown in merged images in b with DAPI signal in blue to indicate the position of nuclei. d–e Representative images of MMC-induced PALB2 foci (d) in cells transfected with either siLacZ or MDC1 siRNAs and quantification (e) of the percentage of HeLa cells with five or more PALB2 foci. PALB2 foci (red) are also shown in merged images in d with DAPI signal in blue. f Immunoblot showing FANCD2 monoubiquitination status in cells treated with a siRNA directed against RNF8 or MDC1. The intensity of the upper monoubiquitinated band divided by the intensity of the lower unubiquitinated band is shown for each lane. g Quantification of the percentage of HeLa cells with five or more ubiquitin foci following transfection with siRNAs and treatment with MMC. Values in c,e,g represent the mean of three independent counts of at least 150 cells each ± standard deviation; * indicates p<0.005.

Article Snippet: Antibodies Primary antibodies utilized for immunofluorescence microscopy and immunoblotting were as follows: FK2 (EMD Millipore, 04-263), FANCD2 (E35) ( Garcia-Higuera et al. 2001 ), PALB2 ( Zhang et al. 2009a ), γH2AX (EMDMillipore, JBW301), RNF8 (Santa Cruz, sc271462), MDC1 (Novus Biologicals, NB100-395), FAAP20 ( Ali et al. 2012 ), RAP80 (Bethyl Laboratory, A300-763A), and anti-HA (Covance, 16B12).

Techniques: Western Blot, Knockdown, Transfection, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 1. Endogenous BRCA1 and ec- topic YFP-BRCA1 form nuclear foci with BARD1 and MDC1. A, endogenous BRCA1 nuclear foci (stained with Ab-4 and Texas Red) co-localize with BARD1 and MDC1 (stained with fluorescein iso- thiocyanate) in T47D cells before and af- ter 15-Gy IR treatment and 4 h of recov- ery. B, YFP-tagged wild-type BRCA1 was transfected into MCF-7 cells and assessed 48 h later for nuclear foci, which also co- localized with BARD1 and MDC1 (stained with Texas Red). Note the redistribution of BRCA1 from few and larger spots in untreated cells to small and more dis- persed foci in IR-treated cells.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 6. Co-localization of BRCA1 BRCT domain fragments and MDC1 in foci. Four YFP-BRCA1 constructs were transiently ex- pressed in MCF-7 cells, which were later irradiated (15 Gy, 4 h of recovery) and co-stained for MDC1 (Texas Red). All ectopic BRCA1 proteins displayed at least partial co-localization with MDC1 foci except the 5382insC mutant, which does not form foci.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Construct, Irradiation, Staining, Mutagenesis

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/elife.61407

Figure Lengend Snippet: Figure 1. Global proteome analysis of cavin3 KO HeLa cells by label-free quantitative proteomics. (A) Z-score for HeLa WT and cavin3 KO cells (replicates Rep. 1–3) showing upregulated proteins (red) and downregulated proteins (blue). (B) Volcano plot showing proteins (red dots) identified by Gene Ontology Biological Process (GOBP) involved in DNA repair. (C) Volcano plot showing DNA repair proteins upregulated in cavin3 KO cells. (D) Volcano plot showing proteins of the BRCA1 A-complex, BRCA1, BRCC3, MDC1, and UBE4A downregulated in cavin3 HeLa KO cells and upregulation of 53BP1 with a heatmap analysis of the expression of each of these proteins in replicate (Rep. 1–3) HeLa WT and cavin3 KO cells. The online version of this article includes the following source data and figure supplement(s) for figure 1:

Article Snippet: DOI: https://doi.org/10.7554/eLife.61407 18 of 35 gamma catenin antibody (Cell Signaling, Cat# 2309, WB 1:3000), rabbit anti-CAV1 antibody (BD Biosciences, Cat# 610060, WB 1:5000), mouse anti-cavin1 antibody (Abmart, China, 1:100 PLA), and rabbit anti-cavin1 antibody were raised as described previously and used for immunofluorescence (Bastiani et al., 2009), rabbit anti-cavin1 antibody (Sigma-Aldrich, Cat# AV36965, RRID:AB 1855947, WB 1:2000), mouse anti-cavin3 antibody (Novus, Cat# H00112464-MO4, PLA 1:200), rabbit anti-cavin3 antibody (Proteintech, Millennium Sciences, Pty, Ltd, Cat# 16250-1-AP, RRID:AB_ 2171897, WB 1:2000, IF 1:300, PLA 1:200), rabbit anti-CHD3 antibody (GeneTex, Sapphire Bioscience, Cat# GTX131779, RRID:AB_2886520, WB 1:500), rabbit anti-DDX21 antibody (Novus, Cat# NBP1-88310, RRID:AB_11027665, WB 1:2000), rabbit anti-EGFR Clone LA22 antibody (Millipore, Cat# 05-104, RRID:AB_11210086, WB 1:4000), mouse-anti-FANCD2 antibody (GeneTex, Cat# GTX116037, RRID:AB2036898, WB 1:500), mouse anti-Flotillin Clone 18 antibody (BD Biosciences, Cat# 610821, RRID:AB_398140, PLA 1:100), mouse anti-GFP antibody (Roche, Cat# 11814460001, RRID:AB_390913, WB 1:4000, PLA 1:300), rabbit anti-Histone H2A.X-Chip Grade (Abcam, Cat# ab20669, RRID:AB_445689, WB 1:1000), rabbit phospho-Histone H2A.X (Ser 139) (20E3) antibody (Cell Signaling Technology, Cat# 9718, RRID:AB_2118009, IF 1:500), rabbit phospho-Histone H2A.X CHIP Grade antibody (Abcam, Cat# ab2893, RRID:AB_303388, WB: 1:3000), rabbit anti-HLTF antibody (Proteintech, Cat# 14286-1-AP, WB 1:2000), rabbit anti-MDC1 antibody (Novus, Cat# 10056657SS, RRID:AB_838567, WB 1:100), sheep anti-Merit40 antibody (R&D Systems, Cat# AF6604SP, RRID:AB_10717577, WB 1:500), rabbit anti-PARP1 antibody (GeneTex, Cat# GTX112864, RRID:AB_11173565, WB 1:1000), mouse anti-PCNA antibody (Millipore, Cat# NA03T, RRID:AB_ 2160357, PLA 1:100), rabbit anti-PGK1 antibody (GeneTex, Cat# GTX107614, RRID:AB_2037666, WB 1:3000), rabbit anti-PKM antibody (GeneTex, Cat# GTX107977, RRID:AB_1951264, WB 1:3000), mouse anti-Rad51 antibody (Novus, Cat# 100-184, RRID:AB_350083, WB 1:1000), rabbit anti-RAP80 D1T6Q antibody (Cell Signaling Technology, Cat# 14466, RRID:AB_2798487, WB 1:1000, IF 1:100), rabbit anti-RNF168 antibody (GeneTex, Cat# GTX118147, RRID:AB_11169617, WB 1:1000), and mouse anti-Tubulin DM1A antibody (Abcam, Cat# ab7291, RRID:AB_2241126, WB 1:4000).

Techniques: Quantitative Proteomics, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/elife.61407

Figure Lengend Snippet: Figure 8. Cavin3-deficient HeLa cells exhibit abolishment of DNA repair. (A) Representative western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. (B) Protein components of the BRCA1 A-complex. Blue-colored circles: proteins downregulated in the label-free quantitative (LFQ) proteomics; yellow-colored circles: proteins not detected in the LFQ proteomics of cavin3 KO cells. (C) Representative western blot analysis of cavin3, BRCA1, pH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1, and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hr chase. Quantitation of protein levels from three independent Figure 8 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.61407 18 of 35 gamma catenin antibody (Cell Signaling, Cat# 2309, WB 1:3000), rabbit anti-CAV1 antibody (BD Biosciences, Cat# 610060, WB 1:5000), mouse anti-cavin1 antibody (Abmart, China, 1:100 PLA), and rabbit anti-cavin1 antibody were raised as described previously and used for immunofluorescence (Bastiani et al., 2009), rabbit anti-cavin1 antibody (Sigma-Aldrich, Cat# AV36965, RRID:AB 1855947, WB 1:2000), mouse anti-cavin3 antibody (Novus, Cat# H00112464-MO4, PLA 1:200), rabbit anti-cavin3 antibody (Proteintech, Millennium Sciences, Pty, Ltd, Cat# 16250-1-AP, RRID:AB_ 2171897, WB 1:2000, IF 1:300, PLA 1:200), rabbit anti-CHD3 antibody (GeneTex, Sapphire Bioscience, Cat# GTX131779, RRID:AB_2886520, WB 1:500), rabbit anti-DDX21 antibody (Novus, Cat# NBP1-88310, RRID:AB_11027665, WB 1:2000), rabbit anti-EGFR Clone LA22 antibody (Millipore, Cat# 05-104, RRID:AB_11210086, WB 1:4000), mouse-anti-FANCD2 antibody (GeneTex, Cat# GTX116037, RRID:AB2036898, WB 1:500), mouse anti-Flotillin Clone 18 antibody (BD Biosciences, Cat# 610821, RRID:AB_398140, PLA 1:100), mouse anti-GFP antibody (Roche, Cat# 11814460001, RRID:AB_390913, WB 1:4000, PLA 1:300), rabbit anti-Histone H2A.X-Chip Grade (Abcam, Cat# ab20669, RRID:AB_445689, WB 1:1000), rabbit phospho-Histone H2A.X (Ser 139) (20E3) antibody (Cell Signaling Technology, Cat# 9718, RRID:AB_2118009, IF 1:500), rabbit phospho-Histone H2A.X CHIP Grade antibody (Abcam, Cat# ab2893, RRID:AB_303388, WB: 1:3000), rabbit anti-HLTF antibody (Proteintech, Cat# 14286-1-AP, WB 1:2000), rabbit anti-MDC1 antibody (Novus, Cat# 10056657SS, RRID:AB_838567, WB 1:100), sheep anti-Merit40 antibody (R&D Systems, Cat# AF6604SP, RRID:AB_10717577, WB 1:500), rabbit anti-PARP1 antibody (GeneTex, Cat# GTX112864, RRID:AB_11173565, WB 1:1000), mouse anti-PCNA antibody (Millipore, Cat# NA03T, RRID:AB_ 2160357, PLA 1:100), rabbit anti-PGK1 antibody (GeneTex, Cat# GTX107614, RRID:AB_2037666, WB 1:3000), rabbit anti-PKM antibody (GeneTex, Cat# GTX107977, RRID:AB_1951264, WB 1:3000), mouse anti-Rad51 antibody (Novus, Cat# 100-184, RRID:AB_350083, WB 1:1000), rabbit anti-RAP80 D1T6Q antibody (Cell Signaling Technology, Cat# 14466, RRID:AB_2798487, WB 1:1000, IF 1:100), rabbit anti-RNF168 antibody (GeneTex, Cat# GTX118147, RRID:AB_11169617, WB 1:1000), and mouse anti-Tubulin DM1A antibody (Abcam, Cat# ab7291, RRID:AB_2241126, WB 1:4000).

Techniques: Western Blot, Quantitation Assay