nb100-395 Search Results


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a Representative immunoblot showing knockdown of <t>MDC1.</t> 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.
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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.

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