ddx1 primary antibodies  (Santa Cruz Biotechnology)

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: The DEAD-box DDX1 is a novel interactor of CDK7. ( A ) Immunoblot analysis with antibodies raised against different TFIIH subunits (XPB and p62 of the core-TFIIH, the bridging factor XPD and the CDK7 and CycH subunits of the CAK), the chromatin (H3), and cytoplasm (Mek2) markers in whole extracts (WCE), 1% triton-soluble (Sol), and chromatin-enriched (Chr) fractions of primary dermal fibroblasts from healthy donors (C3PV and C8PV), PS-TTD (TTD8PV and TTD23PV), or XP (XP26VI and XP15PV) patients with pathogenic variants in ERCC2/XPD gene. Protein quantifications were obtained by normalizing each protein band on the corresponding MEK2 or H3 protein amounts for the soluble and chromatin-enriched fractions, respectively, in order to even out the loading differences. For each TFIIH subunit, the soluble (white bar) and chromatin-bound (black bar) amounts are expressed as a percentage of their sum, indicated as 100%. The graph (bottom panel) includes the data obtained by the immunoblots shown in . The percentage of chromatin-associated proteins in PS-TTD or XP primary dermal fibroblasts is compared with that observed in CTR cells. Bars indicate standard errors (** P < .01, *** P < .001; Student’s t -test). ( B ) Silver staining of proteins co-immunoprecipitated with CDK7 antibodies in the chromatin-enriched (Chr) fraction of MRC5 cells. ( C ) Table list of the novel chromatin-associated CDK7-interacting proteins in MRC5 cells identified through mass spectrometry analysis. Immunoblot analysis of DDX1 protein, the CAK (CDK7 and CycH) and XPD subunits of TFIIH in 1% triton-soluble (sol) and chromatin-enriched fractions (chr) of MRC5 cells before (Input) and after immunoprecipitation (IP) with anti-CDK7 ( D ), anti-DDX1 ( E ), or IgG (D and E) antibodies.

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Western Blot, Silver Staining, Immunoprecipitation, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: DDX1 helicase binds to holo-TFIIH and Pol II. ( A ) In vitro pull-down assay of XPD or the CAK subunits CDK7 and CycH with DDX1 recombinant protein under permissive (100 mM KCl) or restrictive (300 mM KCl) salt conditions. Proteins are visualized by immunoblot analysis with antibodies raised against DDX1, XPD, CDK7, and CycH. The membranes were first hybridized with anti-XPD (*) and subsequently with anti-DDX1. Due to the similar molecular weight of the two proteins, the XPD protein band is still visible in the DDX1 immunoblotting. The input represents 10% of the total protein amount used in each pull-down reaction. In vitro pull-down assays of XPD WT, XPD fragments ( B ), or mutated forms of XPD ( C ) with DDX1 recombinant protein. XPD fragments contain specific functional domains of the protein, as depicted on the XP schematic representation (top). Mutated forms include the Arg112His and Arg722Trp substitutions causative of PS-TTD and the Arg683Trp amino acid change causative of XP. Proteins are visualized by immunoblot analysis with antibodies specific for XPD and DDX1. The input represents 10% of the total protein amount used in each pull-down reaction. ( D ) Immunoblot analysis with antibodies raised against the DDX1 helicase, various TFIIH subunits (XPB and p62 of core-TFIIH, the bridging factor XPD, CDK7, and CycH of the CAK sub-complex), and the RPB1 subunit of Pol II of two-step (TIP) immunoprecipitations performed first with anti-CDK7 and subsequently anti-DDX1 antibodies in 1% triton-soluble (sol) and chromatin-enriched (chr) fractions of control MRC5 cells. As a negative control, the first immunoprecipitation step was performed with IgG antibodies.

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: In Vitro, Pull Down Assay, Recombinant, Western Blot, Molecular Weight, Functional Assay, Control, Negative Control, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: Reduced DDX1 protein amount leads to impaired Pol II-mediated transcription. ( A ) NER efficiency by UDS analysis in MRC5 cells upon gene silencing with either scrambled (CTR) or DDX1 siRNA. The number of individual grains per nuclei was counted. The diagram reports the mean values of three independent experiments. ( B ) Repair efficiency by slot blot analysis of CPD or 6–4PP in untreated MRC5 cells or treated with either DDX1 or control siRNA (CTR) at 0, 1.5, 4.5, 6.5 h after 20 J/m 2 UV-C irradiation. The amount of CPD or 6–4PP has been normalized to the amount of loaded single-strand DNA in at least three independent experiments. No significant differences have been recorded by the Student’s t -test when comparing the mean values of DDX1 silenced cells with the corresponding controls (MRC5 or CTR siRNA). ( C ) Global RNA synthesis by in vivo MRC5 cells labelled with 5-ethynyluridine (EU) and EU-click reaction (red). As a negative control, cells were exposed to Actinomycin D (ACTD) before EU treatment. Nuclei were counterstained with DAPI. The diagram on the left reports the intensity of the EU nuclear fluorescent signal measured by ImageJ in cells from three independent experiments (shown by different colours). The mean value and standard error for each experiment are indicated. ( D ) Immunoblot analysis of DDX1, TFIIH subunits (XPB, p62, XPD, CDK7, and CycH), the α subunit of the basal transcription factor TFIIE and the RPB1 subunit of Pol II in MRC5 whole cell extract transfected with either scrambled (CTR) or DDX1 siRNA (left). The amount of DDX1 and Pol II protein levels was normalized to the amount of β-actin. The diagram reports the mean values of three independent experiments (right). ( E ) Immunoblot analysis with antibodies raised against DDX1, the CDK7 and CycH subunits of TFIIH, the RPB1 subunit of Pol II, and the additional novel CDK7-interactors (NONO, SFPQ, and DHX9) in TIP samples performed first with anti-CDK7 and subsequently anti-DDX1 antibodies in the chromatin-enriched fractions of control MRC5 cells. As a negative control, the first immunoprecipitation step was performed with IgG antibodies (IgG). In all the graphs of the figure, when depicted, bars indicate standard errors (** P < .01, **** P < .0001; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Dot Blot, Control, Irradiation, In Vivo, Negative Control, Western Blot, Transfection, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: Reduced DDX1 protein level leads to increased R-loop amount. ( A ) Slot blot of 0.5 and 1 μg of genomic DNA from MRC5 cells transfected with scrambled control (CTR) or DDX1 siRNA in the absence (−) or presence (+) of RNase H1 and hybridized with antibodies recognizing the RNA/DNA hybrids (S9.6 antibody) or dsDNA (left). The intensity of the bands was measured with ImageJ. The amount of S9.6 signal was normalized to the amount of the loading control dsDNA (right). The values are the mean of at least three independent experiments (* P < .05; Student’s t -test). ( B ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of MRC5 cells treated with scrambled (CTR) or DDX1 siRNA for 120 h. The amount of RNA/DNA hybrids at a, b, c, d, and e positions, indicated as horizontal bars within the β-actin locus (schematic representation on the top), was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least three independent experiments (* P < .05, ** P < .01; Student’s t -test). ( C ) Density plot of DDX1 ChIP-seq peak distribution relative to the gene TSS in Mus musculus . The plot shows the distribution of DDX1 occupancy across 8504 genes, each showing at least one DDX1 ChIP-seq peak within −1000 to +1000 bp of TSS. Peaks were mapped separately to both the plus strand and the minus strand. The x -axis indicates the upstream and downstream distance in bp from the TSS corresponding to position 0 bp. The y -axis indicates the density of DDX1 peaks.

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Dot Blot, Transfection, Control, Real-time Polymerase Chain Reaction, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: DDX1 -dependent R-loop accumulation leads to transcriptional stress. ( A ) Global RNA synthesis by in vivo labelling with 5-ethynyluridine (EU) and EU-click reaction in MRC5 cells 48 h after transfection with DDX1 or scrambled (CTR) siRNA as well as with the plasmid expressing the RNase H1 GFP or the empty vector. Nuclei were counterstained with DAPI. The diagram (top right) reports the intensity of the EU nuclear fluorescent signal measured by ImageJ in cells from two independent experiments (shown by different colours). The mean value and standard errors of each experiment are indicated (*** P < 0.001; Student’s t -test). ( B ) Immunoblot analysis with antibodies raised against DDX1, XPD, γH2AX, and ORC2 in the chromatin-enriched fraction of cells transfected with either scrambled (CTR) or DDX1 siRNA (left). The amount of DDX1, XPD, and γH2AX protein levels was first normalized to the amount of the chromatin loading control ORC2 and then expressed as fold increased relative to the corresponding protein amount in CTR siRNA. The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (** P < .01, *** P < .001; Student’s t -test). ( C ) Immunoblot analysis with antibodies raised against DDX1, γH2AX, and γTub in MRC5 cells transfected with DDX1 or scrambled (CTR) siRNA as well as with the plasmid expressing the RNase H1 GFP (+) or the empty vector (−) (left). The amount of DDX1 and γH2AX protein levels was normalized to the amount of the corresponding γTub loading control and reported as arbitrary units (au). The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (* P < .05, *** P < .001; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: TFIIH plays a role in R-loop processing. ( A ) Immunoblot analysis with antibodies raised against the RPB1 subunit of Pol II and various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK) in MRC5 whole cell extracts transfected with scrambled control (CTR) or XPD siRNA. Protein levels were normalized to the amount of γ-tubulin and reported as arbitrary units (au). The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (* P < .05, ** P < .01, *** P < .001; Student’s t -test). ( B ) Slot blot of 0.5 and 1 μg of genomic DNA from MRC5 cells transfected with scrambled control (CTR) or XPD siRNAs in the absence (−) or presence (+) of RNase H1 and hybridized with antibodies recognizing the RNA/DNA hybrids (S9.6 antibody) or dsDNA (left panel). The intensity of the bands was measured with ImageJ. The amount of S9.6 signal was normalized to the amount of the loading control, the dsDNA signal (right panel). The values are the mean of at least three independent experiments. Bars indicate the standard error (* P < .05; Student’s t -test). ( C ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of MRC5 cells treated with scrambled control (CTR) or XPD siRNA for 72 h. The amount of RNA/DNA hybrids at the a, b, c, d, and e positions of the β-actin locus was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least three independent experiments. Bars indicate the standard errors (* P < .05, ** P < .01; Student’s t -test). ( D ) Immunoblot analysis of XPD and the CDK7 subunits of TFIIH, NONO, SFPQ, and DDX1 proteins in the chromatin-enriched fractions of MRC5 cells transfected with scrambled control (CTR) or XPD siRNAs before (Input) and after immunoprecipitation (IP) with anti-CDK7 or IgG antibodies. Two independent IPs are shown (upper and lower left panels). In the cells treated with XPD -siRNA, the amount of co-immunoprecipitated proteins has been normalized to the amount of the corresponding immunoprecipitated CDK7 and expressed as fold increased relative to the sample CTR siRNA (right panel). The values are the mean of at least three independent experiments. When depicted, bars indicate standard errors (* P < .05; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Western Blot, Transfection, Control, Dot Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: PS-TTD cells exhibit altered TFIIH-DDX1 interaction and impaired R-loop processing. ( A ) Immunoblot analysis with antibodies raised against various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK), DDX1, NONO, SFPQ, and the RPB1 subunit of Pol II in 1% triton-soluble (sol) and chromatin-enriched (chr) fractions of MRC5, TTD2GL, and XP102LO cells before (Input) and after immunoprecipitation with anti-CDK7 antibodies. The amount of co-immunoprecipitated DDX1, NONO, or SFPQ proteins was normalized to the amount of immunoprecipitated CDK7 (right panel). Bars indicate standard errors (* P < .05; ** P < .01; *** P < .001; Student’s t -test). ( B ) Immunoblot analysis with antibodies raised against various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK), DDX1, NONO, SFPQ, and the RPB1 subunit of Pol II in 1% triton-soluble (sol) and chromatin-enriched (chr) fractions of control (C3PV), TTD23PV, and XP26VI primary dermal fibroblasts before (Input) and after immunoprecipitation (IP) with anti-CDK7 or IgG antibodies. The amount of co-immunoprecipitated DDX1, NONO, and SFPQ was normalized to the amount of immunoprecipitated CDK7. The graph (right panel) also includes data collected from the samples shown in . Bars indicate standard errors (** P < .01; Student’s t -test). ( C ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of primary dermal fibroblasts from PS-TTD with pathogenic variants in ERCC2/XPD (TTD8PV, TTD12PV, and TTD23PV) or in ERCC3/XPB (TTD6VI), from XP with pathogenic variants in ERCC2/XPD (XP15PV and XP49PV), or healthy individuals (CTR, C3PV, and C5PV). The amount of RNA/DNA hybrids at the a, b, c, d, and e positions of the ACTB locus was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least two independent experiments. Bars indicate standard errors (* P < .05, ** P < .01, *** P < .001; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Western Blot, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction

Journal: Retrovirology

Article Title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

doi: 10.1186/s12977-014-0121-9

Figure Lengend Snippet: Figure 2 Interactions of DDX1, DDX3 and DDX17 with Nullbasic in vivo. HEK293T cells were transfected with either empty vector (pcDNA 3.1) or plasmid expressing NB-FLAG. The FLAG-tagged proteins and their binding partners were immunoprecipated with anti-FLAG beads. Total cell lystes and immunoprecipated proteins were separated by SDS-PAGE and target proteins were detected using anit-FLAG, anti-DDX1, anti-DDX3 and anti-DDX17 antibodies. The anti-CDK9 antibody was used to detect endogenous CDK9 as a positive control for NB interaction. The figure is representative of 3 independent experiments.

Article Snippet: Protein lysates were also immunoprecipitated with mouse DDX1 primary antibody (Santa Cruz) conjugated to protein G magnetic beads (Sigma-Aldrich) as recommended by manufacture and eluted with SDS-PAGE sample buffer.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Binding Assay, SDS Page, Positive Control

Journal: Retrovirology

Article Title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

doi: 10.1186/s12977-014-0121-9

Figure Lengend Snippet: Figure 3 Overexpression of DDX1 in HeLa cells rescues the Rev/RRE-dependent reporter gene expression suppressed by Nullbasic. A. Schematic maps of pGag-RRE and pGag-CTE plasmids. B. Left: HeLa cells were transfected with appropriate expression plasmids, including pGag-RRE (500 ng), pRSV-Rev (10 ng), Nullbasic (NB, 500 ng), HA-DDX1(1000 ng), HA-DDX3 (1000 ng), DDX17 (1000 ng) and empty plasmid (pcDNA3-HA, 1000 g). Right: HeLa cells were transfected pGag-CTE (500 ng) with pRSV-Rev (10 ng), Nullbasic (NB) (500 ng), HA-DDX1(1000 ng) or empty vector (pcDNA3-HA, 1000 ng). A pcDNA3 plasmid was used to normalize the total amount of transfected plasmids and a luciferase expression plasmid was included to monitor transfection efficiency. After 24 h transfection, the cellular lysates were collected for assay of HIV-1 capsid level and luciferase activity. The p24 production was normalized to the luciferase reporter activity and expressed as a percentage relative to cells transfected with pGag-RRE with pRSV-Rev (left) or pGag-CTE with pRSV-Rev (right). Columns represent the means and standard deviations of transfections performed in triplicate. The experiment was performed 3 times with similar results.

Article Snippet: Protein lysates were also immunoprecipitated with mouse DDX1 primary antibody (Santa Cruz) conjugated to protein G magnetic beads (Sigma-Aldrich) as recommended by manufacture and eluted with SDS-PAGE sample buffer.

Techniques: Over Expression, Gene Expression, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Retrovirology

Article Title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

doi: 10.1186/s12977-014-0121-9

Figure Lengend Snippet: Figure 4 Nullbasic alters the subcellular localization of DDX1. A. HeLa cells were transfected with empty vector alone (pcDNA3.1, row 1), MYC-Rev alone (row 2), Nullbasic (NB)-mCherry alone (row 3 a) or MYC-Rev with NB-mCherry (row 4). Fixed cells were immunostained with anti-MYC (green) and anti-DDX1 (magenta, upper panels) and were visualized alone with NB-mCherry (red) by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Rev panel with DDX1 (row 1, 2, and 4) and the NB-mCherry panel with DDX1 panel (row 3). Images are representative of at least five fields selected randomly from three independent experiments. B. Quantification of nuclear/cytoplasmic fluorescence ratios(Fn/c) for DDX1as described in Methods. Mean of Fn/c ± SD were calculated from 60 HeLa cells from 3 independent experiments.

Article Snippet: Protein lysates were also immunoprecipitated with mouse DDX1 primary antibody (Santa Cruz) conjugated to protein G magnetic beads (Sigma-Aldrich) as recommended by manufacture and eluted with SDS-PAGE sample buffer.

Techniques: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Staining

Journal: Retrovirology

Article Title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

doi: 10.1186/s12977-014-0121-9

Figure Lengend Snippet: Figure 5 Overexpression of DDX1 in HeLa cells restores Rev nucleolar localization disrupted by Nullbasic. HeLa cells were transfected with HA-DDX1 alone (row 1), MYC-Rev with HA-DDX1 (row 2). Nullbasic (NB)-mCherry with HA-DDX1 (row 3) or MYC-Rev with HA-DDX1 and NB-mCherry (row 4). Fixed cells were immunostained with anti-MYC (green) and anti-HA (magenta) antibodies and were visualized alone with NB-mCherry (red) by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the MYC-Rev panel with the HA-DDX1 panels (row 1, 2, and 4) and the NB-mCherry panel with HA-DDX1 panel (row 3). Images are representative of at least five fields selected randomly from three independent experiments.

Article Snippet: Protein lysates were also immunoprecipitated with mouse DDX1 primary antibody (Santa Cruz) conjugated to protein G magnetic beads (Sigma-Aldrich) as recommended by manufacture and eluted with SDS-PAGE sample buffer.

Techniques: Over Expression, Transfection, Fluorescence, Microscopy, Staining

Journal: Retrovirology

Article Title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

doi: 10.1186/s12977-014-0121-9

Figure Lengend Snippet: Figure 6 DDX1 directly interacts with Nullbasic in vitro. The Octet Red system was used to measure binding events between Nullbasic and DDX1. A. BLI sensograms. Biotinylated recombinant Nullbasic-FLAG-V5-6 × His was bound to a streptavidin biosensor and applied to solutions containing 3.3 nM (green), 10 nM (light blue), 30 nM (red) or 90 nM (dark blue) of human recombinant Myc-DDK-tagged DDX1. B. The probe was also exposed to DDX5 solutions of the same concentration or with BSA at 90 nM as negative controls. The BLI experiment was repeated 3 times with similar results and a representative sensogram is shown.

Article Snippet: Protein lysates were also immunoprecipitated with mouse DDX1 primary antibody (Santa Cruz) conjugated to protein G magnetic beads (Sigma-Aldrich) as recommended by manufacture and eluted with SDS-PAGE sample buffer.

Techniques: In Vitro, Binding Assay, Recombinant, Concentration Assay

Journal: Retrovirology

Article Title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

doi: 10.1186/s12977-014-0121-9

Figure Lengend Snippet: Figure 7 DDX1 forms different protein complexes with Nullbasic and Rev. A. HEK293T cells were transfected with either empty vector (pcDNA 3.1) alone, Nullbasic (NB)-FLAG alone, Rev alone or co-transfect NB-FLAG with Rev as indicated. Endogenous DDX1, NB-FLAG and their binding partners were immunoprecipated with anti-DDX1 and anti-FLAG beads. Cell lystes and immunoprecipated proteins were separated by SDS-PAGE and target proteins were detected using anti-FLAG, anti-DDX1 and anti-Rev antibodies. B. HEK293T cells were transfected with empty vector or Tat-FLAG as indicated. Target proteins were detected using anti-FLAG, anti-DDX1 after immunoprecipitation using anti-DDX1, anti-FLAG beads or with beads alone. The figures are representative of 3 independent experiments.

Article Snippet: Protein lysates were also immunoprecipitated with mouse DDX1 primary antibody (Santa Cruz) conjugated to protein G magnetic beads (Sigma-Aldrich) as recommended by manufacture and eluted with SDS-PAGE sample buffer.

Techniques: Transfection, Plasmid Preparation, Binding Assay, SDS Page, Immunoprecipitation