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α nucks1  (Novus Biologicals)
90
Novus Biologicals α nucks1
<t>NUCKS1</t> deficiency leads to an increase in RAD54 foci upon exposure of cells to DNA-damaging agents. (A) Schematic of the primary structure of the 243–amino acid human NUCKS1 protein with two nuclear localization sequences (NLS1 and NLS2; ) and one DNA binding domain (DBD; ). (B) Representative micrographs to show pan-nuclear expression of NUCKS1 in HeLa cells, a clonal isolate of HeLa cells transfected with a nontargeting sgRNA (Ctrl-1), two clonal isolates of HeLa cells that are KO for NUCKS1 (KO-1 and KO-2), and one clonal isolate of KO-1 cells ectopically expressing NUCKS1. Scale bar, 10 µm. (C) Western blots of fractionated nuclear extracts (nuclear soluble and chromatin bound). The signals for PARP1 and Histone H3 serve as loading and fractionation controls, respectively. (D) In response to 8 Gy γ-rays, NUCKS1 KO cells form more RAD54 foci. KO-1+N, KO-1 cells ectopically expressing NUCKS1. Bars represent the mean from two to seven independent experiments (symbols). Error bars, ±1 SEM; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (E) In response to MMC treatment, NUCKS1 KO cells form more RAD54 foci. Bars represent the mean from two independent experiments (symbols). Error bars, ±1 SEM; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (F) Results from clonogenic survival assays to show that HeLa cells depleted for RAD54, KO-1 cells, and KO-1 cells depleted for RAD54 display the same sensitivity to the cytotoxic effects of MMC. Data points represent the mean from three independent experiments. Error bars, ± 1SEM. ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (G) Representative Western blots from whole cell extracts to show RAD54 knockdown in cells used in F. si-control, nondepleting control siRNA. siRAD54, RAD54-depleting siRNA. PARP1, loading control.
α Nucks1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/α nucks1/product/Novus Biologicals
Average 90 stars, based on 1 article reviews
α nucks1 - by Bioz Stars, 2026-02
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anti nucks1 antibody  (Novus Biologicals)
90
Novus Biologicals anti nucks1 antibody
Interaction between HIV-1 Tat and <t>NUCKS1.</t> (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204. The transformants were grown on an SD plate lacking adenosine and uracil for 48 h. Murine p53 and SV40 large T antigens were used as a positive control. Two panels show yeast colonies showing the interaction of Tat and NUCKS1. (B) Ectopically expressed Tat interacts with ectopically expressed NUCKS1 in HEK293 cells. Two micrograms of the Flag–Tat, Flag-Vpr and Flag-Nef expression vector were cotransfected with the V5–NUCKS1 expression vector into HEK293 cells cultured in 100 mm plates. Forty-eight hours after transfection, cell lysates were immunoprecipitated with an anti-Flag monoclonal antibody (M2). Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-V5 monoclonal antibody. The Flag- or V5-tagged pcDNA3 plasmid was added to equalize the total amounts of DNA. (C) Coimmunoprecipitation assay between endogenous NUCKS1 and ectopically expressed Tat protein. The lysate from Tat-expressing HEK293T cells was immunoprecipitated with anti-Flag and -Pol II antibodies, and the interaction was assessed with Western blotting using anti-NUCKS1 or anti-Flag antibody.
Anti Nucks1 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti nucks1 antibody/product/Novus Biologicals
Average 90 stars, based on 1 article reviews
anti nucks1 antibody - by Bioz Stars, 2026-02
90/100 stars
  Buy from Supplier

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NUCKS1 deficiency leads to an increase in RAD54 foci upon exposure of cells to DNA-damaging agents. (A) Schematic of the primary structure of the 243–amino acid human NUCKS1 protein with two nuclear localization sequences (NLS1 and NLS2; ) and one DNA binding domain (DBD; ). (B) Representative micrographs to show pan-nuclear expression of NUCKS1 in HeLa cells, a clonal isolate of HeLa cells transfected with a nontargeting sgRNA (Ctrl-1), two clonal isolates of HeLa cells that are KO for NUCKS1 (KO-1 and KO-2), and one clonal isolate of KO-1 cells ectopically expressing NUCKS1. Scale bar, 10 µm. (C) Western blots of fractionated nuclear extracts (nuclear soluble and chromatin bound). The signals for PARP1 and Histone H3 serve as loading and fractionation controls, respectively. (D) In response to 8 Gy γ-rays, NUCKS1 KO cells form more RAD54 foci. KO-1+N, KO-1 cells ectopically expressing NUCKS1. Bars represent the mean from two to seven independent experiments (symbols). Error bars, ±1 SEM; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (E) In response to MMC treatment, NUCKS1 KO cells form more RAD54 foci. Bars represent the mean from two independent experiments (symbols). Error bars, ±1 SEM; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (F) Results from clonogenic survival assays to show that HeLa cells depleted for RAD54, KO-1 cells, and KO-1 cells depleted for RAD54 display the same sensitivity to the cytotoxic effects of MMC. Data points represent the mean from three independent experiments. Error bars, ± 1SEM. ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (G) Representative Western blots from whole cell extracts to show RAD54 knockdown in cells used in F. si-control, nondepleting control siRNA. siRAD54, RAD54-depleting siRNA. PARP1, loading control.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: NUCKS1 deficiency leads to an increase in RAD54 foci upon exposure of cells to DNA-damaging agents. (A) Schematic of the primary structure of the 243–amino acid human NUCKS1 protein with two nuclear localization sequences (NLS1 and NLS2; ) and one DNA binding domain (DBD; ). (B) Representative micrographs to show pan-nuclear expression of NUCKS1 in HeLa cells, a clonal isolate of HeLa cells transfected with a nontargeting sgRNA (Ctrl-1), two clonal isolates of HeLa cells that are KO for NUCKS1 (KO-1 and KO-2), and one clonal isolate of KO-1 cells ectopically expressing NUCKS1. Scale bar, 10 µm. (C) Western blots of fractionated nuclear extracts (nuclear soluble and chromatin bound). The signals for PARP1 and Histone H3 serve as loading and fractionation controls, respectively. (D) In response to 8 Gy γ-rays, NUCKS1 KO cells form more RAD54 foci. KO-1+N, KO-1 cells ectopically expressing NUCKS1. Bars represent the mean from two to seven independent experiments (symbols). Error bars, ±1 SEM; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (E) In response to MMC treatment, NUCKS1 KO cells form more RAD54 foci. Bars represent the mean from two independent experiments (symbols). Error bars, ±1 SEM; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (F) Results from clonogenic survival assays to show that HeLa cells depleted for RAD54, KO-1 cells, and KO-1 cells depleted for RAD54 display the same sensitivity to the cytotoxic effects of MMC. Data points represent the mean from three independent experiments. Error bars, ± 1SEM. ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (G) Representative Western blots from whole cell extracts to show RAD54 knockdown in cells used in F. si-control, nondepleting control siRNA. siRAD54, RAD54-depleting siRNA. PARP1, loading control.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Binding Assay, Expressing, Transfection, Western Blot, Fractionation, Knockdown, Control

Data supporting . (A) Schematic of the human NUCKS1 gene with its seven exons, the locations of the targeting sequences of the sgRNAs (see also ) and of PCR primers (P1–P8; see also ) used in this study. Note the NUCKS1 gene is located on the minus strand of the human reference genome. Here, NUCKS1 has been flipped 180° for simplicity. (B) Schematics of the three NUCKS1 KO alleles detected in HeLa NUCKS1 KO-1 cells. PCR products obtained by amplifying genomic DNA from NUCKS1 KO-1 cells with P1-P3 and P4-P2, respectively, were isolated and amplified by topoisomerase-based cloning (Invitrogen) and sequenced. A fourth NUCKS1 KO allele could not be detected, potentially due to absence or due to a large deletion encompassing the binding sequences for P1 and/or P2. (C) Representative agarose gels obtained after amplifying genomic DNA from different cell lines with primers P1-P3 and P4-P2, as indicated. (D) Representative micrographs of nuclear RAD54 foci obtained for the results presented in . Scale bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: Data supporting . (A) Schematic of the human NUCKS1 gene with its seven exons, the locations of the targeting sequences of the sgRNAs (see also ) and of PCR primers (P1–P8; see also ) used in this study. Note the NUCKS1 gene is located on the minus strand of the human reference genome. Here, NUCKS1 has been flipped 180° for simplicity. (B) Schematics of the three NUCKS1 KO alleles detected in HeLa NUCKS1 KO-1 cells. PCR products obtained by amplifying genomic DNA from NUCKS1 KO-1 cells with P1-P3 and P4-P2, respectively, were isolated and amplified by topoisomerase-based cloning (Invitrogen) and sequenced. A fourth NUCKS1 KO allele could not be detected, potentially due to absence or due to a large deletion encompassing the binding sequences for P1 and/or P2. (C) Representative agarose gels obtained after amplifying genomic DNA from different cell lines with primers P1-P3 and P4-P2, as indicated. (D) Representative micrographs of nuclear RAD54 foci obtained for the results presented in . Scale bars, 10 µm.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Isolation, Amplification, Cloning, Binding Assay

Data supporting . (A) Western blots to show that the amount of nuclear-soluble and chromatin-bound RAD54 is not different between control cells (HeLa and KO-1+N) and NUCKS1 KO cells (KO-1 and KO-2). (B) Results from flow cytometry showing two-color fluorescence of cell cycle profiles of the cells used in . Y axis, EdU; x axis, SYTOX. (C) Western blot to show loss of NUCKS1 expression in HT1080 NUCKS1 KO cells. QM, loading control. (D) HT1080 NUCKS1 KO cells form more nuclei with RAD54 foci than HT1080 control cells (Ctrl, clonal isolate of HT1080 cells transfected with a nontargeting sgRNA). Data are from one experiment. (E) Representative micrographs of nuclear RAD54 foci obtained for the results presented in . Scale bars, 10 µm. (F) Results from flow cytometry showing two-color fluorescence of cell cycle profiles of the cells used in . Y axis, EdU; x axis; SYTOX. (G) Results from clonogenic survival assays to show that NUCKS1 KO HeLa cells (KO-1 and KO-2) are more sensitive to the cytotoxic effects of MMC than Ctrl-1 cells. Data points represent the mean from three independent experiments. Error bars, ±1 SD. *, P < 0.05; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: Data supporting . (A) Western blots to show that the amount of nuclear-soluble and chromatin-bound RAD54 is not different between control cells (HeLa and KO-1+N) and NUCKS1 KO cells (KO-1 and KO-2). (B) Results from flow cytometry showing two-color fluorescence of cell cycle profiles of the cells used in . Y axis, EdU; x axis, SYTOX. (C) Western blot to show loss of NUCKS1 expression in HT1080 NUCKS1 KO cells. QM, loading control. (D) HT1080 NUCKS1 KO cells form more nuclei with RAD54 foci than HT1080 control cells (Ctrl, clonal isolate of HT1080 cells transfected with a nontargeting sgRNA). Data are from one experiment. (E) Representative micrographs of nuclear RAD54 foci obtained for the results presented in . Scale bars, 10 µm. (F) Results from flow cytometry showing two-color fluorescence of cell cycle profiles of the cells used in . Y axis, EdU; x axis; SYTOX. (G) Results from clonogenic survival assays to show that NUCKS1 KO HeLa cells (KO-1 and KO-2) are more sensitive to the cytotoxic effects of MMC than Ctrl-1 cells. Data points represent the mean from three independent experiments. Error bars, ±1 SD. *, P < 0.05; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Western Blot, Control, Flow Cytometry, Fluorescence, Expressing, Transfection

NUCKS1 deficiency leads to a transient delay in RAD51 foci formation, and NUCKS1 and RAD54 functionally interact. (A) The formation of IR-induced RAD51 foci is delayed in KO-1 and KO-2 cells. Bars represent the mean from two to three independent experiments (symbols). Error bars, ±1 SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (B) RAD54 knockdown impairs RAD51 foci formation in NUCKS1-expressing cells (HeLa and KO-1+N), but not in NUCKS1 KO cells (KO-1 and KO-2). Bars represent the mean from two independent experiments (symbols). Error bars, ±1 SEM; **, P < 0.01; ***, P < 0.001; ns, not significant; two-way ANOVA analysis. (C i) Schematic of the D-loop reaction with naked pBluescript II SK(−) plasmid DNA. (C ii) NUCKS1 (60, 120, 600, or 1,200 nM) does not promote the RAD51-mediated D-loop reaction (lanes 2–5). RAD54 (100, 200, or 400 nM) promotes the RAD51-mediated D-loop reaction (lanes 6–8). NUCKS1 (60 or 600 nM) promotes the RAD51–RAD54-mediated D-loop reaction (compare lanes 9 and 10 with lane 6). (C iii) Quantification of the results from three independent experiments. Bars are the means. Error bars, ±1 SD. *, P < 0.05; **, P < 0.01; multiple t test analysis. (D i) Schematic of the D-loop reaction with chromatinized pBluescript II SK(−) plasmid DNA. (D ii) RAD54 (100 or 200 nM) promotes the RAD51-mediated D-loop reaction on chromatinized DNA (lanes 2 and 3). NUCKS1 (600 nM) promotes the RAD51-mediated D-loop reaction in the presence of RAD54 (lane 5), but not in the absence of RAD54 or ATP (lanes 7 and 6, respectively). (D iii) Quantification of the results from three independent experiments. Bars are the means. Error bars, ±1 SD. **, P < 0.01; ns, not significant; multiple t test analysis. (E i) Thin-layer chromatogram to test for the ATPase activity on dsDNA (125 ng) of RAD54 (100 nM), RAD54+NUCKS1 (100 nM + 300 nM), and NUCKS1 alone (300 and 600 nM). (E ii) Quantification of the results. Data are from one experiment only. (F i) Thin-layer chromatogram to show that NUCKS1 (150 and 300 nM) stimulates the ATPase activity of RAD54 on dsDNA. (F ii) Quantification of the results. Data points are the mean from three independent experiments. Error bars, ±1 SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; multiple t test analysis.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: NUCKS1 deficiency leads to a transient delay in RAD51 foci formation, and NUCKS1 and RAD54 functionally interact. (A) The formation of IR-induced RAD51 foci is delayed in KO-1 and KO-2 cells. Bars represent the mean from two to three independent experiments (symbols). Error bars, ±1 SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. (B) RAD54 knockdown impairs RAD51 foci formation in NUCKS1-expressing cells (HeLa and KO-1+N), but not in NUCKS1 KO cells (KO-1 and KO-2). Bars represent the mean from two independent experiments (symbols). Error bars, ±1 SEM; **, P < 0.01; ***, P < 0.001; ns, not significant; two-way ANOVA analysis. (C i) Schematic of the D-loop reaction with naked pBluescript II SK(−) plasmid DNA. (C ii) NUCKS1 (60, 120, 600, or 1,200 nM) does not promote the RAD51-mediated D-loop reaction (lanes 2–5). RAD54 (100, 200, or 400 nM) promotes the RAD51-mediated D-loop reaction (lanes 6–8). NUCKS1 (60 or 600 nM) promotes the RAD51–RAD54-mediated D-loop reaction (compare lanes 9 and 10 with lane 6). (C iii) Quantification of the results from three independent experiments. Bars are the means. Error bars, ±1 SD. *, P < 0.05; **, P < 0.01; multiple t test analysis. (D i) Schematic of the D-loop reaction with chromatinized pBluescript II SK(−) plasmid DNA. (D ii) RAD54 (100 or 200 nM) promotes the RAD51-mediated D-loop reaction on chromatinized DNA (lanes 2 and 3). NUCKS1 (600 nM) promotes the RAD51-mediated D-loop reaction in the presence of RAD54 (lane 5), but not in the absence of RAD54 or ATP (lanes 7 and 6, respectively). (D iii) Quantification of the results from three independent experiments. Bars are the means. Error bars, ±1 SD. **, P < 0.01; ns, not significant; multiple t test analysis. (E i) Thin-layer chromatogram to test for the ATPase activity on dsDNA (125 ng) of RAD54 (100 nM), RAD54+NUCKS1 (100 nM + 300 nM), and NUCKS1 alone (300 and 600 nM). (E ii) Quantification of the results. Data are from one experiment only. (F i) Thin-layer chromatogram to show that NUCKS1 (150 and 300 nM) stimulates the ATPase activity of RAD54 on dsDNA. (F ii) Quantification of the results. Data points are the mean from three independent experiments. Error bars, ±1 SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; multiple t test analysis.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Knockdown, Expressing, Plasmid Preparation, Activity Assay

Data supporting . (A) Representative micrographs of nuclear RAD51 foci obtained for the results presented in . Scale bars, 10 µm. (B) Representative Western blots to show RAD54 knockdown in cells used in . (C) Representative micrographs of nuclear RAD51 foci obtained for the results presented in . Scale bars, 10 µm. (D) SDS-PAGE to show purified human RAD54-FLAG and MBP-RAD51AP1-(His) 6 . (E) SDS-PAGE to show purified human RAD51. (F) SDS-PAGE to show purified human GST-NUCKS1 and (His) 6 -RAD51AP1-FLAG. (G) Naked and chromatinized pBluescript II SK(−) DNA after micrococcal nuclease (MNase) digest and deproteinization to show periodicity of nucleosomes and nucleosomal repeat lengths (lanes 5–7). (H) Western blot to show lost expression of RAD54 in RAD54 KO HeLa cells. MW, molecular weight.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: Data supporting . (A) Representative micrographs of nuclear RAD51 foci obtained for the results presented in . Scale bars, 10 µm. (B) Representative Western blots to show RAD54 knockdown in cells used in . (C) Representative micrographs of nuclear RAD51 foci obtained for the results presented in . Scale bars, 10 µm. (D) SDS-PAGE to show purified human RAD54-FLAG and MBP-RAD51AP1-(His) 6 . (E) SDS-PAGE to show purified human RAD51. (F) SDS-PAGE to show purified human GST-NUCKS1 and (His) 6 -RAD51AP1-FLAG. (G) Naked and chromatinized pBluescript II SK(−) DNA after micrococcal nuclease (MNase) digest and deproteinization to show periodicity of nucleosomes and nucleosomal repeat lengths (lanes 5–7). (H) Western blot to show lost expression of RAD54 in RAD54 KO HeLa cells. MW, molecular weight.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Western Blot, Knockdown, SDS Page, Purification, Expressing, Molecular Weight

NUCKS1 and RAD54 physically interact. (A) SDS-PAGE to show the interaction between GST-NUCKS1 and RAD54 in a GST pull-down assay (lane 3). RAD54 does not precipitate nonspecifically (lane 6). S, supernatant containing unbound proteins; W, wash; E, SDS eluate of glutathione resin. (B) Western blot to show the interaction between FLAG-RAD54 and NUCKS1 precipitated by anti-FLAG M2 affinity resin (lane 4). NUCKS1 does not precipitate nonspecifically (lane 8). S, supernatant containing unbound proteins; W, wash 1 and 2, respectively; E, eluate. (C) Western blot to show the interaction between FLAG-RAD54, NUCKS1, and RAD51 precipitated by anti-FLAG M2 affinity resin (lane 3). RAD51 does not precipitate nonspecifically (lane 4). (D) Western blot to show that endogenous NUCKS1 interacts weakly with full-length RAD54 (lane 6), the RAD54 lobe1+2 fragment (lane 9), and the C-terminal domain of RAD54 (lane 10), strongly with the RAD54 lobe 1 fragment (lane 8), and not with the RAD54 N-terminal domain (lane 7). IP, immunoprecipitation.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: NUCKS1 and RAD54 physically interact. (A) SDS-PAGE to show the interaction between GST-NUCKS1 and RAD54 in a GST pull-down assay (lane 3). RAD54 does not precipitate nonspecifically (lane 6). S, supernatant containing unbound proteins; W, wash; E, SDS eluate of glutathione resin. (B) Western blot to show the interaction between FLAG-RAD54 and NUCKS1 precipitated by anti-FLAG M2 affinity resin (lane 4). NUCKS1 does not precipitate nonspecifically (lane 8). S, supernatant containing unbound proteins; W, wash 1 and 2, respectively; E, eluate. (C) Western blot to show the interaction between FLAG-RAD54, NUCKS1, and RAD51 precipitated by anti-FLAG M2 affinity resin (lane 3). RAD51 does not precipitate nonspecifically (lane 4). (D) Western blot to show that endogenous NUCKS1 interacts weakly with full-length RAD54 (lane 6), the RAD54 lobe1+2 fragment (lane 9), and the C-terminal domain of RAD54 (lane 10), strongly with the RAD54 lobe 1 fragment (lane 8), and not with the RAD54 N-terminal domain (lane 7). IP, immunoprecipitation.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: SDS Page, Pull Down Assay, Western Blot, Immunoprecipitation

Data supporting . (A) Representative micrographs to show EdU repair patches (red) in nuclei with RAD54 foci (green) at 8 h after 8 Gy γ-irradiation. Scale bar, 10 µm. (B) DNA repair synthesis after γ-irradiation (as determined by EdU incorporation in G2-phase cells) is abrogated in both HeLa and NUCKS1 KO-1 cells after RAD54 knockdown. (C) Representative micrographs obtained for the data shown in B. Scale bars, 10 µm. S, S-phase cells; G2, G2-phase cells. (D) DNA repair synthesis after γ-irradiation is slightly delayed in NUCKS1 KO-1 cells. Bars represent the means from one to three independent experiments (symbols). Error bars, ±1 SEM; ns, not significant (P > 0.185 for all data points); two-way ANOVA analysis; MW, molecular weight.

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: Data supporting . (A) Representative micrographs to show EdU repair patches (red) in nuclei with RAD54 foci (green) at 8 h after 8 Gy γ-irradiation. Scale bar, 10 µm. (B) DNA repair synthesis after γ-irradiation (as determined by EdU incorporation in G2-phase cells) is abrogated in both HeLa and NUCKS1 KO-1 cells after RAD54 knockdown. (C) Representative micrographs obtained for the data shown in B. Scale bars, 10 µm. S, S-phase cells; G2, G2-phase cells. (D) DNA repair synthesis after γ-irradiation is slightly delayed in NUCKS1 KO-1 cells. Bars represent the means from one to three independent experiments (symbols). Error bars, ±1 SEM; ns, not significant (P > 0.185 for all data points); two-way ANOVA analysis; MW, molecular weight.

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Irradiation, Knockdown, Molecular Weight

NUCKS1 deficiency negatively impacts HR protein complex formation. (A) RAD54–RAD51 PLA foci in sham-irradiated and irradiated HeLa and NUCKS1 KO-1 cells. Symbols, average values for 250 nuclei from two to three independent experiments each. Bars, overall means. Error bars, ±1 SD. ****, P < 0.0001; one-way ANOVA analysis. (B) RAD54–RAD51AP1 PLA foci in sham-irradiated and irradiated HeLa and NUCKS1 KO-1 cells. Symbols, average values for 250 nuclei from two to three independent experiments each. Bars, overall means. Error bars, ±1 SD. ****, P < 0.0001; one-way ANOVA analysis. (C) Western blots to show that increasing amounts of RAD51AP1 can interfere with the RAD54–RAD51 complex. Interaction between FLAG-RAD54 and RAD51 precipitated by anti-FLAG M2 affinity resin (lane 1). Interaction between FLAG-RAD54 and MBP-RAD51AP1 precipitated by anti-FLAG M2 affinity resin (lane 2). Increasing the amounts of RAD51AP1 competes with the interaction between FLAG-RAD54 and RAD51 (lanes 3 and 4, respectively). RAD51 does not precipitate nonspecifically; excessive MBP-RAD51AP1 (2 µM) does precipitate nonspecifically on anti-FLAG M2 affinity resin (lane 5). Blue, relative signal intensities for RAD51. (D) Western blot to show that purified NUCKS1 and RAD51AP1 interact (lane 3). NUCKS1 does not precipitate nonspecifically on anti-FLAG M2 resin (lane 2). (E) Preincubation of NUCKS1 with RAD51AP1 diminishes the amount of either protein precipitated in anti-FLAG RAD54 complexes (lane 5). Neither NUCKS1 nor RAD51AP1 precipitates nonspecifically on anti-FLAG M2 resin (lane 2). RAD51AP1 interacts with RAD54 (lane 3). NUCKS1 interacts with RAD54 (lane 4). Blue, relative signal intensities for NUCKS1 and RAD51AP1. (F) Knockdown of RAD51AP1 (here, AP1) in NUCKS1 KO-1 cells reduces IR-induced RAD54 foci. Bars represent the means from two to four independent experiments (symbols). Error bars, ±1 SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. si-control, nondepleting negative control siRNA. si-AP1, RAD51AP1-depleting siRNA (see ). (G) Results from clonogenic survival assays to show that KO-1 cells with XRCC4 knockdown are more sensitive to the cytotoxic effects of γ-rays than HeLa cells with XRCC4 knockdown. Data points are the mean from three independent experiments. Error bars, ±1 SEM. ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. si-control, nondepleting negative control siRNA; si-XRCC4, XRCC4-depleting siRNA (see ).

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: NUCKS1 deficiency negatively impacts HR protein complex formation. (A) RAD54–RAD51 PLA foci in sham-irradiated and irradiated HeLa and NUCKS1 KO-1 cells. Symbols, average values for 250 nuclei from two to three independent experiments each. Bars, overall means. Error bars, ±1 SD. ****, P < 0.0001; one-way ANOVA analysis. (B) RAD54–RAD51AP1 PLA foci in sham-irradiated and irradiated HeLa and NUCKS1 KO-1 cells. Symbols, average values for 250 nuclei from two to three independent experiments each. Bars, overall means. Error bars, ±1 SD. ****, P < 0.0001; one-way ANOVA analysis. (C) Western blots to show that increasing amounts of RAD51AP1 can interfere with the RAD54–RAD51 complex. Interaction between FLAG-RAD54 and RAD51 precipitated by anti-FLAG M2 affinity resin (lane 1). Interaction between FLAG-RAD54 and MBP-RAD51AP1 precipitated by anti-FLAG M2 affinity resin (lane 2). Increasing the amounts of RAD51AP1 competes with the interaction between FLAG-RAD54 and RAD51 (lanes 3 and 4, respectively). RAD51 does not precipitate nonspecifically; excessive MBP-RAD51AP1 (2 µM) does precipitate nonspecifically on anti-FLAG M2 affinity resin (lane 5). Blue, relative signal intensities for RAD51. (D) Western blot to show that purified NUCKS1 and RAD51AP1 interact (lane 3). NUCKS1 does not precipitate nonspecifically on anti-FLAG M2 resin (lane 2). (E) Preincubation of NUCKS1 with RAD51AP1 diminishes the amount of either protein precipitated in anti-FLAG RAD54 complexes (lane 5). Neither NUCKS1 nor RAD51AP1 precipitates nonspecifically on anti-FLAG M2 resin (lane 2). RAD51AP1 interacts with RAD54 (lane 3). NUCKS1 interacts with RAD54 (lane 4). Blue, relative signal intensities for NUCKS1 and RAD51AP1. (F) Knockdown of RAD51AP1 (here, AP1) in NUCKS1 KO-1 cells reduces IR-induced RAD54 foci. Bars represent the means from two to four independent experiments (symbols). Error bars, ±1 SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. si-control, nondepleting negative control siRNA. si-AP1, RAD51AP1-depleting siRNA (see ). (G) Results from clonogenic survival assays to show that KO-1 cells with XRCC4 knockdown are more sensitive to the cytotoxic effects of γ-rays than HeLa cells with XRCC4 knockdown. Data points are the mean from three independent experiments. Error bars, ±1 SEM. ***, P < 0.001; ****, P < 0.0001; ns, not significant; two-way ANOVA analysis. si-control, nondepleting negative control siRNA; si-XRCC4, XRCC4-depleting siRNA (see ).

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Irradiation, Western Blot, Purification, Knockdown, Control, Negative Control

Model to explain how NUCKS1 regulates spatiotemporal events in the HR reaction. In undamaged cells, RAD54 and RAD51AP1 are associated with NUCKS1 to prevent the inappropriate association of RAD54 with RAD51AP1. Upon exposure of cells to IR, RAD54 is able to (1) stabilize the presynaptic RAD51 filament (which does not require RAD54 ATPase activity) and (2) stimulate the RAD54 ATPase for strand invasion and formation of heteroduplex DNA. In the absence of NUCKS1, RAD54 is inappropriately complexed with RAD51AP1. This complex negatively impacts the timely formation of RAD51 foci (see ), as it delays the interaction between RAD54 and RAD51 that is required for a stable RAD51-ssDNA nucleoprotein filament ( ; ; ; ). RAD51AP1 also promotes strand invasion ( ; ).

Journal: The Journal of Cell Biology

Article Title: NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

doi: 10.1083/jcb.201911049

Figure Lengend Snippet: Model to explain how NUCKS1 regulates spatiotemporal events in the HR reaction. In undamaged cells, RAD54 and RAD51AP1 are associated with NUCKS1 to prevent the inappropriate association of RAD54 with RAD51AP1. Upon exposure of cells to IR, RAD54 is able to (1) stabilize the presynaptic RAD51 filament (which does not require RAD54 ATPase activity) and (2) stimulate the RAD54 ATPase for strand invasion and formation of heteroduplex DNA. In the absence of NUCKS1, RAD54 is inappropriately complexed with RAD51AP1. This complex negatively impacts the timely formation of RAD51 foci (see ), as it delays the interaction between RAD54 and RAD51 that is required for a stable RAD51-ssDNA nucleoprotein filament ( ; ; ; ). RAD51AP1 also promotes strand invasion ( ; ).

Article Snippet: The working concentrations of the primary antibodies used were as follows: α-RAD51 (H-92; Santa Cruz Biotechnology; 1:1,000), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:1,000), α-RAD51AP1 (NBP2-13197; Novus Biologicals; 1:1,000), and α-NUCKS1 (ab84710; Novus Biologicals; 1:300).

Techniques: Activity Assay

Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204. The transformants were grown on an SD plate lacking adenosine and uracil for 48 h. Murine p53 and SV40 large T antigens were used as a positive control. Two panels show yeast colonies showing the interaction of Tat and NUCKS1. (B) Ectopically expressed Tat interacts with ectopically expressed NUCKS1 in HEK293 cells. Two micrograms of the Flag–Tat, Flag-Vpr and Flag-Nef expression vector were cotransfected with the V5–NUCKS1 expression vector into HEK293 cells cultured in 100 mm plates. Forty-eight hours after transfection, cell lysates were immunoprecipitated with an anti-Flag monoclonal antibody (M2). Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-V5 monoclonal antibody. The Flag- or V5-tagged pcDNA3 plasmid was added to equalize the total amounts of DNA. (C) Coimmunoprecipitation assay between endogenous NUCKS1 and ectopically expressed Tat protein. The lysate from Tat-expressing HEK293T cells was immunoprecipitated with anti-Flag and -Pol II antibodies, and the interaction was assessed with Western blotting using anti-NUCKS1 or anti-Flag antibody.

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204. The transformants were grown on an SD plate lacking adenosine and uracil for 48 h. Murine p53 and SV40 large T antigens were used as a positive control. Two panels show yeast colonies showing the interaction of Tat and NUCKS1. (B) Ectopically expressed Tat interacts with ectopically expressed NUCKS1 in HEK293 cells. Two micrograms of the Flag–Tat, Flag-Vpr and Flag-Nef expression vector were cotransfected with the V5–NUCKS1 expression vector into HEK293 cells cultured in 100 mm plates. Forty-eight hours after transfection, cell lysates were immunoprecipitated with an anti-Flag monoclonal antibody (M2). Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-V5 monoclonal antibody. The Flag- or V5-tagged pcDNA3 plasmid was added to equalize the total amounts of DNA. (C) Coimmunoprecipitation assay between endogenous NUCKS1 and ectopically expressed Tat protein. The lysate from Tat-expressing HEK293T cells was immunoprecipitated with anti-Flag and -Pol II antibodies, and the interaction was assessed with Western blotting using anti-NUCKS1 or anti-Flag antibody.

Article Snippet: The expression level of NUCKS1 was analyzed by Western blotting using anti-NUCKS1 antibody (Novus Biologicals).

Techniques: Expressing, Plasmid Preparation, Clone Assay, cDNA Library Assay, Transformation Assay, Positive Control, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

NUCKS1-mediated Tat activation on HIV-1 LTR. (A) One hundred nanograms of the Flag–Tat and/or 3 μg of the NUCKS1 expression plasmid was transfected with 200 ng of the HIV-1 LTR-driven luciferase reporter (pGL3–LTR–Luc) and 20 ng of pCMV–LacZ as a transfection control into HeLa cells. At 24 h after transfection, luciferase activity was measured and normalized to β-galactosidase activity. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared with the Flag-Tat transfected only. Expression levels of NUCKS1 and Tat were assessed with Western blotting. (B) Luciferase activity was assessed in HeLa cells 24 h after cotransfection of pGL3–LTR–Luc (200 ng), Flag–Tat (100 ng), and increasing amounts of V5–NUCKS1 (0, 0.5, 1, 2, and 3 μg), together with 20 ng of pCMV–LacZ as a transfection control. The data are expressed as mean ± SD (n = 3). *, P < 0.01 and **, P < 0.05, as compared with the Flag-Tat transfected only (C) Knockdown of NUCKS1 was performed with three siRNAs against NUCKS1 mRNA. HeLa cells were transfected with control siRNA or three NUCKS1 siRNAs. At 48 h after transfection, knockdown of NUCKS1 by siRNA was assessed by Western blotting using anti-NUCKS1, anti-Flag and anti-β-actin antibodies as a loading control. (D) NUCKS1-engaed Tat activity was assessed by the luciferase assay combined with the knockdown experiment. HeLa cells were transfected with control siRNA or three NUCKS1 siRNAs. Twenty-four hours after transfection, cells were transfected further with pGL3–LTR–Luc, pCMV– LacZ , and Flag–Tat. The luciferase assay was conducted as described in A. The data was expressed as mean ± SD (n = 3). *, P < 0.01 as compared with the cells transfected with control siRNA.

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: NUCKS1-mediated Tat activation on HIV-1 LTR. (A) One hundred nanograms of the Flag–Tat and/or 3 μg of the NUCKS1 expression plasmid was transfected with 200 ng of the HIV-1 LTR-driven luciferase reporter (pGL3–LTR–Luc) and 20 ng of pCMV–LacZ as a transfection control into HeLa cells. At 24 h after transfection, luciferase activity was measured and normalized to β-galactosidase activity. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared with the Flag-Tat transfected only. Expression levels of NUCKS1 and Tat were assessed with Western blotting. (B) Luciferase activity was assessed in HeLa cells 24 h after cotransfection of pGL3–LTR–Luc (200 ng), Flag–Tat (100 ng), and increasing amounts of V5–NUCKS1 (0, 0.5, 1, 2, and 3 μg), together with 20 ng of pCMV–LacZ as a transfection control. The data are expressed as mean ± SD (n = 3). *, P < 0.01 and **, P < 0.05, as compared with the Flag-Tat transfected only (C) Knockdown of NUCKS1 was performed with three siRNAs against NUCKS1 mRNA. HeLa cells were transfected with control siRNA or three NUCKS1 siRNAs. At 48 h after transfection, knockdown of NUCKS1 by siRNA was assessed by Western blotting using anti-NUCKS1, anti-Flag and anti-β-actin antibodies as a loading control. (D) NUCKS1-engaed Tat activity was assessed by the luciferase assay combined with the knockdown experiment. HeLa cells were transfected with control siRNA or three NUCKS1 siRNAs. Twenty-four hours after transfection, cells were transfected further with pGL3–LTR–Luc, pCMV– LacZ , and Flag–Tat. The luciferase assay was conducted as described in A. The data was expressed as mean ± SD (n = 3). *, P < 0.01 as compared with the cells transfected with control siRNA.

Article Snippet: The expression level of NUCKS1 was analyzed by Western blotting using anti-NUCKS1 antibody (Novus Biologicals).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Transfection, Luciferase, Control, Activity Assay, Western Blot, Cotransfection, Knockdown

Mechanism linked to NUCKS1-mediated Tat activation. (A) HeLa cells were transfected with the V5–NUCKS1 expression plasmid. At 24 h after transfection, some of the cells were treated with PMA (50 ng/ml) for 15 min before harvest, and the cells were harvested. Nucleus and cytoplasm from the cells were fractionated. NF-κB activity was assessed by Western blotting using anti-p65, -Iκb, and -NUCKS1 antibodies. The nuclear and cytoplasmic fractions were evaluated using anti-lamin B and -tubulin antibodies, respectively. (B) NUCKS1-mediated NF-κB promoter activity was assessed by luciferase assay. HeLa cells transfected with NF-κB luciferase reporter plasmid (500 ng), V5–NUCKS1 (3 μg), and pCMV– LacZ (20 ng). The luciferase assay was performed 24 h after transfection. PMA (50 ng/ml) treatment was used as a positive control. The data are expressed as mean ± SD (n = 3). (C) HeLa cells were cotransfected with the Flag–Tat and V5–NUCKS1 expression plasmids. Twenty-four hours after transfection, the nucleus and cytoplasm were fractionated, and the subcellular localization of Tat and NUCKS1 was assessed with Western blotting using anti-Flag, anti-V5, -lamin B, and -α-tubulin antibodies, respectively. (D) HeLa cells were transfected with Flag–Tat, V5–NUCKS1, or both. Two days after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. Cyc T1–Tat interaction was assessed with Western blotting using anti-Cyclin T1 and anti-Flag antibodies, respectively.

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Mechanism linked to NUCKS1-mediated Tat activation. (A) HeLa cells were transfected with the V5–NUCKS1 expression plasmid. At 24 h after transfection, some of the cells were treated with PMA (50 ng/ml) for 15 min before harvest, and the cells were harvested. Nucleus and cytoplasm from the cells were fractionated. NF-κB activity was assessed by Western blotting using anti-p65, -Iκb, and -NUCKS1 antibodies. The nuclear and cytoplasmic fractions were evaluated using anti-lamin B and -tubulin antibodies, respectively. (B) NUCKS1-mediated NF-κB promoter activity was assessed by luciferase assay. HeLa cells transfected with NF-κB luciferase reporter plasmid (500 ng), V5–NUCKS1 (3 μg), and pCMV– LacZ (20 ng). The luciferase assay was performed 24 h after transfection. PMA (50 ng/ml) treatment was used as a positive control. The data are expressed as mean ± SD (n = 3). (C) HeLa cells were cotransfected with the Flag–Tat and V5–NUCKS1 expression plasmids. Twenty-four hours after transfection, the nucleus and cytoplasm were fractionated, and the subcellular localization of Tat and NUCKS1 was assessed with Western blotting using anti-Flag, anti-V5, -lamin B, and -α-tubulin antibodies, respectively. (D) HeLa cells were transfected with Flag–Tat, V5–NUCKS1, or both. Two days after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. Cyc T1–Tat interaction was assessed with Western blotting using anti-Cyclin T1 and anti-Flag antibodies, respectively.

Article Snippet: The expression level of NUCKS1 was analyzed by Western blotting using anti-NUCKS1 antibody (Novus Biologicals).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Luciferase, Positive Control, Immunoprecipitation

Close association between NUCKS1 and Tat binding to TAR RNA. HeLa cells transiently transfected with HIV-1 LTR (A and C) or TZM-bl cells containing an integrated HIV-1 LTR (B and D) were transfected with control siRNA or three individual NUCKS1 siRNAs. At 24 h after knockdown, these cells were transiently transfected with the Flag–Tat expression plasmid and cultured for an additional 24 h. Cells were cross-linked with formaldehyde, and chromatin immunoprecipitation was performed using IgG control or anti-Flag antibody (A and B) or anti-Cyclin T1 (C and D) . Quantitative real-time PCR was performed; the data were normalized to the IgG control antibody and are expressed as fold change of Tat binding activity compared with control siRNA-treated samples. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to the cells transfected with control siRNA.

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Close association between NUCKS1 and Tat binding to TAR RNA. HeLa cells transiently transfected with HIV-1 LTR (A and C) or TZM-bl cells containing an integrated HIV-1 LTR (B and D) were transfected with control siRNA or three individual NUCKS1 siRNAs. At 24 h after knockdown, these cells were transiently transfected with the Flag–Tat expression plasmid and cultured for an additional 24 h. Cells were cross-linked with formaldehyde, and chromatin immunoprecipitation was performed using IgG control or anti-Flag antibody (A and B) or anti-Cyclin T1 (C and D) . Quantitative real-time PCR was performed; the data were normalized to the IgG control antibody and are expressed as fold change of Tat binding activity compared with control siRNA-treated samples. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to the cells transfected with control siRNA.

Article Snippet: The expression level of NUCKS1 was analyzed by Western blotting using anti-NUCKS1 antibody (Novus Biologicals).

Techniques: Binding Assay, Transfection, Control, Knockdown, Expressing, Plasmid Preparation, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay

Key role of NUCKS1 in viral production and replication. (A-B) In the single-round replication assay, HeLa cells were transfected with control siRNA or three individual NUCKS1 siRNAs. One day later, the cells were transfected with pNL4-3 (A) or pAD8 (B) proviral DNA. Two days after siRNA transfection, the virus-containing supernatants were harvested every day for 5 days. The amount of virion in the supernatant was measured by HIV-1 p24 ELISA. (C) In the viral replication assay, Jurkat and A3.01 cells were transfected with control siRNA or three individual NUCKS1 siRNAs. One day later, the cells were infected with pNL4-3 virus. Five days after infection, the virus-containing supernatants were harvested, and then the amount of virions in the supernatant was measured by HIV-1 p24 ELISA. The data are expressed as mean ± SD (n = 3). * , P < 0.01 as compared with the cells transfected with control siRNA. The lower panels show the intracellular p24 levels.

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Key role of NUCKS1 in viral production and replication. (A-B) In the single-round replication assay, HeLa cells were transfected with control siRNA or three individual NUCKS1 siRNAs. One day later, the cells were transfected with pNL4-3 (A) or pAD8 (B) proviral DNA. Two days after siRNA transfection, the virus-containing supernatants were harvested every day for 5 days. The amount of virion in the supernatant was measured by HIV-1 p24 ELISA. (C) In the viral replication assay, Jurkat and A3.01 cells were transfected with control siRNA or three individual NUCKS1 siRNAs. One day later, the cells were infected with pNL4-3 virus. Five days after infection, the virus-containing supernatants were harvested, and then the amount of virions in the supernatant was measured by HIV-1 p24 ELISA. The data are expressed as mean ± SD (n = 3). * , P < 0.01 as compared with the cells transfected with control siRNA. The lower panels show the intracellular p24 levels.

Article Snippet: The expression level of NUCKS1 was analyzed by Western blotting using anti-NUCKS1 antibody (Novus Biologicals).

Techniques: Transfection, Control, Virus, Enzyme-linked Immunosorbent Assay, Viral Replication Assay, Infection

Requirement of NUCKS1 for reactivation of provirus from HIV-1 latently infected cells. (A) Heatmap of mRNA expression of NUCKS1 and other Tat-interacting factors. (B) Expression level of endogenous NUCKS1 in HIV-1 latent cells (ACH-2 and J1.1) and their parent cell lines (A3.01 and Jurkat) were assessed with Western blotting using anti-NUCKS1 and anti-β-actin antibodies. The protein level of NUCKS1 was calculated by densitometry (Image Gauge Version 4.0, FujiFilm) (C) Normal and latently infected cells were treated with PMA (50 ng/ml) for indicated time. PMA-treated cell lysates were assessed by Western blotting using indicated antibodies. The levels of HIV-1 p24 were measured using an HIV-1 p24 ELISA kit. The data are expressed as mean ± SD (n = 3). (D) mRNA-sequencing analysis. The RPKM (Reads Per Kilobase of exon model per Million aligned tags) was calculated for each transcript. The Y-axis represents the log2 fold changes between the control and PMA-treated ACH-2 cells. The right panel shows the expression level of NUCKS1 mRNA by RT-PCR. (E) ACH-2 and J1.1 cells were electrophorated with V5 or V5-NUCKS1. Three days later, the amount of virion in the supernatant was measured by HIV-1 p24 ELISA kit. The right panel shows the level of NUCKS1 expression by Western blotting. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to cells transfected with empty vector. (F-G) ACH-2 (F) and J1.1 cells (G) were electroporated with control or three individual NUCKS1 siRNAs. At 48 h after knockdown, the cells were treated with PMA (50 ng/ml) for 24 h and the levels of HIV-1 p24 were measured using an HIV-1 p24 ELISA kit. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to the cells transfected with control siRNA.

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Requirement of NUCKS1 for reactivation of provirus from HIV-1 latently infected cells. (A) Heatmap of mRNA expression of NUCKS1 and other Tat-interacting factors. (B) Expression level of endogenous NUCKS1 in HIV-1 latent cells (ACH-2 and J1.1) and their parent cell lines (A3.01 and Jurkat) were assessed with Western blotting using anti-NUCKS1 and anti-β-actin antibodies. The protein level of NUCKS1 was calculated by densitometry (Image Gauge Version 4.0, FujiFilm) (C) Normal and latently infected cells were treated with PMA (50 ng/ml) for indicated time. PMA-treated cell lysates were assessed by Western blotting using indicated antibodies. The levels of HIV-1 p24 were measured using an HIV-1 p24 ELISA kit. The data are expressed as mean ± SD (n = 3). (D) mRNA-sequencing analysis. The RPKM (Reads Per Kilobase of exon model per Million aligned tags) was calculated for each transcript. The Y-axis represents the log2 fold changes between the control and PMA-treated ACH-2 cells. The right panel shows the expression level of NUCKS1 mRNA by RT-PCR. (E) ACH-2 and J1.1 cells were electrophorated with V5 or V5-NUCKS1. Three days later, the amount of virion in the supernatant was measured by HIV-1 p24 ELISA kit. The right panel shows the level of NUCKS1 expression by Western blotting. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to cells transfected with empty vector. (F-G) ACH-2 (F) and J1.1 cells (G) were electroporated with control or three individual NUCKS1 siRNAs. At 48 h after knockdown, the cells were treated with PMA (50 ng/ml) for 24 h and the levels of HIV-1 p24 were measured using an HIV-1 p24 ELISA kit. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to the cells transfected with control siRNA.

Article Snippet: The expression level of NUCKS1 was analyzed by Western blotting using anti-NUCKS1 antibody (Novus Biologicals).

Techniques: Infection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Knockdown