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Addgene inc trim33 plasmid

FIGURE 2. KAP1 inhibits RNA viralinduced IFN-b activation. (A) Luciferase analysis of IFN-b reporter activation in HEK293T cells trans- fected with IFN-b reporter plasmid with empty control, TRIM28 (KAP1), TRIM24, <t>TRIM33,</t> or TRIM66 expression plasmid and then infected with SeV for 12 h. (B) Luciferase analysis of IFN-b reporter activation in HEK293T cells transfected with IFN-b reporter plasmid, together with empty control plasmid or KAP1 expression plasmids, and then infected with VSV for 12 h. (C) Western blot analysis of KAP1 expression in mouse PMs from WT or CKO mice. Data shown are means ± SD [n 5 6 in (A), and n 5 5 in (B)]. All experiments were repeated three times. **p < 0.01, ***p < 0.001, by Student t test.

Trim33 Plasmid, supplied by Addgene inc, 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/product/trim33+gfp/pm34497149-65-0-10?v=Addgene+inc
Average 90 stars, based on 1 article reviews

trim33 plasmid - by Bioz Stars, 2026-07

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1) Product Images from "KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5."

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.2100342

Figure Legend Snippet: FIGURE 2. KAP1 inhibits RNA viralinduced IFN-b activation. (A) Luciferase analysis of IFN-b reporter activation in HEK293T cells trans- fected with IFN-b reporter plasmid with empty control, TRIM28 (KAP1), TRIM24, TRIM33, or TRIM66 expression plasmid and then infected with SeV for 12 h. (B) Luciferase analysis of IFN-b reporter activation in HEK293T cells transfected with IFN-b reporter plasmid, together with empty control plasmid or KAP1 expression plasmids, and then infected with VSV for 12 h. (C) Western blot analysis of KAP1 expression in mouse PMs from WT or CKO mice. Data shown are means ± SD [n 5 6 in (A), and n 5 5 in (B)]. All experiments were repeated three times. **p < 0.01, ***p < 0.001, by Student t test.

Techniques Used: Activation Assay, Luciferase, Plasmid Preparation, Control, Expressing, Infection, Transfection, Western Blot

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Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 2. KAP1 inhibits RNA viralinduced IFN-b activation. (A) Luciferase analysis of IFN-b reporter activation in HEK293T cells trans- fected with IFN-b reporter plasmid with empty control, TRIM28 (KAP1), TRIM24, TRIM33, or TRIM66 expression plasmid and then infected with SeV for 12 h. (B) Luciferase analysis of IFN-b reporter activation in HEK293T cells transfected with IFN-b reporter plasmid, together with empty control plasmid or KAP1 expression plasmids, and then infected with VSV for 12 h. (C) Western blot analysis of KAP1 expression in mouse PMs from WT or CKO mice. Data shown are means ± SD [n 5 6 in (A), and n 5 5 in (B)]. All experiments were repeated three times. **p < 0.01, ***p < 0.001, by Student t test.

Article Snippet: TRIM33 plasmid (65398) and TRIM66 plasmid (65400) were purchased from Addgene and subcloned into the V5 tag to generate V5-TRIM33 and V5-TRIM66.

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Control, Expressing, Infection, Transfection, Western Blot

FIGURE 1. TRIM33 interacts with ALC1 upon induction of DNA damage. A, HEK293 cells expressing either vector or FLAG-ALC1 were treated with either vehicle or bleomycin, subjected to immunoprecipitation using FLAG beads, and then processed by LC-MS/MS. The relative peptide counts for each condition are shown for ALC1, PARP1, APLF, histone H2B, Ku70, and TRIM33. As shown, TRIM33 peptides were identified only in the bleomycin-treated samples. B, endogenous interaction of TRIM33 and ALC1 upon hydroxyurea (HU) and bleomycin treatment. DNase-treated nuclear extracts from untreated (Un) cells or cells treated with HU (3 mM, 3 h) or bleomycin (300uM, 1 h) were immunoprecipitated with anti-TRIM33 antibody. Immunoprecipitates were processed for Western blotting (WB) using antibodies to TRIM33 and ALC1 (top two panels, IP). Aliquots of nuclear extract were also directly processed for Western blotting with these antibodies (bottom two panels, Inputs).

Journal: Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE 1. TRIM33 interacts with ALC1 upon induction of DNA damage. A, HEK293 cells expressing either vector or FLAG-ALC1 were treated with either vehicle or bleomycin, subjected to immunoprecipitation using FLAG beads, and then processed by LC-MS/MS. The relative peptide counts for each condition are shown for ALC1, PARP1, APLF, histone H2B, Ku70, and TRIM33. As shown, TRIM33 peptides were identified only in the bleomycin-treated samples. B, endogenous interaction of TRIM33 and ALC1 upon hydroxyurea (HU) and bleomycin treatment. DNase-treated nuclear extracts from untreated (Un) cells or cells treated with HU (3 mM, 3 h) or bleomycin (300uM, 1 h) were immunoprecipitated with anti-TRIM33 antibody. Immunoprecipitates were processed for Western blotting (WB) using antibodies to TRIM33 and ALC1 (top two panels, IP). Aliquots of nuclear extract were also directly processed for Western blotting with these antibodies (bottom two panels, Inputs).

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Western Blot

FIGURE 2. TRIM33 participates in DNA damage response. A, Schematics of the TRIM33 constructs showing relevant domains and the mutant constructs used. B, HeLa cells were treated with UV laser scissors and processed for IF using antibodies to H2AX (green) and TRIM33 (red) at the times indicated. C, quantitation of TRIM33 dynamics at the site of UV laser-induced DNA breaks (mean S.D. of at least 20 cells). D, HeLa cells transfected with FLAG-WtTRIM33, TRIM33CA, TRIM33PHD, TRIM33 PHD(AAA), or TRIM33Bromo were treated with UV laser scissors and, after 10 min, processed for IF using antibodies to FLAG and H2AX. E, quantitation of TRIM33 constructs at UV laser-induced DNA breaks (mean S.D. of at least 20 cells).

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE 2. TRIM33 participates in DNA damage response. A, Schematics of the TRIM33 constructs showing relevant domains and the mutant constructs used. B, HeLa cells were treated with UV laser scissors and processed for IF using antibodies to H2AX (green) and TRIM33 (red) at the times indicated. C, quantitation of TRIM33 dynamics at the site of UV laser-induced DNA breaks (mean S.D. of at least 20 cells). D, HeLa cells transfected with FLAG-WtTRIM33, TRIM33CA, TRIM33PHD, TRIM33 PHD(AAA), or TRIM33Bromo were treated with UV laser scissors and, after 10 min, processed for IF using antibodies to FLAG and H2AX. E, quantitation of TRIM33 constructs at UV laser-induced DNA breaks (mean S.D. of at least 20 cells).

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Construct, Mutagenesis, Quantitation Assay, Transfection

FIGURE 3. TRIM33 knockdown results in DNA damage sensitivity. A, HeLa cells treated with control shRNA, TRIM33 shRNA1, TRIM33 shRNA2, and TRIM33 siRNA were exposed to increasing concentrations of bleomycin. Relative cell counts measured by MTS assay, normalized to no treatment, were performed on day 3 and were plotted. p 0.005 for control versus TRIM33 shRNA or siRNA. Relative expression of TRIM33 and tubulin is shown. B, HeLa cells treated with controlshRNA,TRIM33shRNA,orTRIM33shRNAcellscomplementedwithWtTRIM33wereexposedtoincreasingconcentrationsofbleomycin,andrelativecell counts were measured as above. The Western blot analysis shows levels of TRIM33 and tubulin. C, whole cell extracts from control and TRIM33 sh2-treated cells were processed for Western blotting using antibodies to the indicated proteins. D, TRIM33 localization to DNA damage is not dependent on ATM, ATR, or DNA-dependent protein kinase (DNApk). HeLa cells treated with vehicle or ATM inhibitor (ATMi) (KU-55933), GM18366 (ATR mutant), and M059J (DNApk/) cells were subject to laser scissor-induced DNA damage. Cells were fixed after 10 min and processed for IF using antibodies to H2AX (green) and TRIM33 (red). E, quantitation of TRIM33 at sites of DNA damage is shown. Each data point is the mean S.D. of at least 20 cells.

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE 3. TRIM33 knockdown results in DNA damage sensitivity. A, HeLa cells treated with control shRNA, TRIM33 shRNA1, TRIM33 shRNA2, and TRIM33 siRNA were exposed to increasing concentrations of bleomycin. Relative cell counts measured by MTS assay, normalized to no treatment, were performed on day 3 and were plotted. p 0.005 for control versus TRIM33 shRNA or siRNA. Relative expression of TRIM33 and tubulin is shown. B, HeLa cells treated with controlshRNA,TRIM33shRNA,orTRIM33shRNAcellscomplementedwithWtTRIM33wereexposedtoincreasingconcentrationsofbleomycin,andrelativecell counts were measured as above. The Western blot analysis shows levels of TRIM33 and tubulin. C, whole cell extracts from control and TRIM33 sh2-treated cells were processed for Western blotting using antibodies to the indicated proteins. D, TRIM33 localization to DNA damage is not dependent on ATM, ATR, or DNA-dependent protein kinase (DNApk). HeLa cells treated with vehicle or ATM inhibitor (ATMi) (KU-55933), GM18366 (ATR mutant), and M059J (DNApk/) cells were subject to laser scissor-induced DNA damage. Cells were fixed after 10 min and processed for IF using antibodies to H2AX (green) and TRIM33 (red). E, quantitation of TRIM33 at sites of DNA damage is shown. Each data point is the mean S.D. of at least 20 cells.

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Knockdown, Control, shRNA, MTS Assay, Expressing, Western Blot, Mutagenesis, Quantitation Assay

FIGURE4.LocalizationofTRIM33toDNAbreaksisdependentuponPARPandALC1.A,PAR(toptwopanels)andTRIM33(bottomtwopanels)werelocalized by IF in untreated (Un) and in cells pretreated with 1 M PARPi (Pi) ABT-888 for 1 h. B, quantitation of PAR and TRIM33 colocalization with H2AX at sites of laser scissors.*,p0.005.C,Parp1/orParp1/mouseembryonicfibroblastsweretreatedwithlaserscissors,andH2AXandTRIM33werelocalizedbyIF.Images are shown at identical magnification. D, quantitation of PAR and TRIM33 colocalization with H2AX at sites of laser scissors. *, p 0.005. E, APLF, WtALC1, C1 (ALC1 macro domain only), and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with 32P-labeled PAR. F, TRIM33 localization to DNA breaks is ALC1-dependent. U2OS cells stably expressing control sh, ALC1sh, or cells expressing ALC1 sh were reconstituted with WT ALC1, KR (kinase dead) and DA (PAR binding mutant) were analyzed. All cells were subjected to UV laser scissor-induced DNA breaks. After 10 min, cell were fixed, and IF was performed using antibodies to H2AX and TRIM33. G, Western blot analyses showing levels of ALC1 and TRIM33 in U2OS cells expressing control and ALC1 shRNA and different constructs of ALC1 in ALC1sh cells. H, quantitation of relative intensity of TRIM33 at sites of DNA damage. Each data point is mean S.D. of at least 20 cells. *, p 0.005.

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE4.LocalizationofTRIM33toDNAbreaksisdependentuponPARPandALC1.A,PAR(toptwopanels)andTRIM33(bottomtwopanels)werelocalized by IF in untreated (Un) and in cells pretreated with 1 M PARPi (Pi) ABT-888 for 1 h. B, quantitation of PAR and TRIM33 colocalization with H2AX at sites of laser scissors.*,p0.005.C,Parp1/orParp1/mouseembryonicfibroblastsweretreatedwithlaserscissors,andH2AXandTRIM33werelocalizedbyIF.Images are shown at identical magnification. D, quantitation of PAR and TRIM33 colocalization with H2AX at sites of laser scissors. *, p 0.005. E, APLF, WtALC1, C1 (ALC1 macro domain only), and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with 32P-labeled PAR. F, TRIM33 localization to DNA breaks is ALC1-dependent. U2OS cells stably expressing control sh, ALC1sh, or cells expressing ALC1 sh were reconstituted with WT ALC1, KR (kinase dead) and DA (PAR binding mutant) were analyzed. All cells were subjected to UV laser scissor-induced DNA breaks. After 10 min, cell were fixed, and IF was performed using antibodies to H2AX and TRIM33. G, Western blot analyses showing levels of ALC1 and TRIM33 in U2OS cells expressing control and ALC1 shRNA and different constructs of ALC1 in ALC1sh cells. H, quantitation of relative intensity of TRIM33 at sites of DNA damage. Each data point is mean S.D. of at least 20 cells. *, p 0.005.

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Quantitation Assay, Membrane, Incubation, Labeling, Stable Transfection, Expressing, Control, Binding Assay, Mutagenesis, Western Blot, shRNA, Construct

FIGURE 5. TRIM33 dynamically interacts with ALC1 in a PARP-dependent manner. A, HeLa cells were untreated (Un) or treated with IR or UV light, with or without PARP inhibitor (PARPi), and DNase-treated nuclear extracts were prepared at 5 and 10 min. TRIM33 IP was performed, followed by Western blotting (WB) using the indicated antibodies. IP, immunoprecipitation; No Ab, no antibody. B, quantitation of ALC1 interaction with TRIM33. The plot shows the ratio of the signal of ALC1 coimmunoprecipitation to ALC1 input. C, The FLAG WtALC1, ALC1K77R, and ALC1D723A mutants were expressed in 293 cells and subjected to UV irradiation. Protein extracts were prepared after 5 min and immunoprecipitated with anti-FLAG antibodies, followed by Western blotting using antibody against TRIM33 and FLAG. D, PAR-bound ALC1 does not bind TRIM33 in vitro. WtALC1, KR (ATPase dead) and DA (PAR binding mutant) ALC1 mutants and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with PAR, washed, and then incubated with purified TRIM33. Membranes were then processed for Western blot analysis with antibodies to PAR (top panel) or antibody to TRIM33 (bottom panel).

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE 5. TRIM33 dynamically interacts with ALC1 in a PARP-dependent manner. A, HeLa cells were untreated (Un) or treated with IR or UV light, with or without PARP inhibitor (PARPi), and DNase-treated nuclear extracts were prepared at 5 and 10 min. TRIM33 IP was performed, followed by Western blotting (WB) using the indicated antibodies. IP, immunoprecipitation; No Ab, no antibody. B, quantitation of ALC1 interaction with TRIM33. The plot shows the ratio of the signal of ALC1 coimmunoprecipitation to ALC1 input. C, The FLAG WtALC1, ALC1K77R, and ALC1D723A mutants were expressed in 293 cells and subjected to UV irradiation. Protein extracts were prepared after 5 min and immunoprecipitated with anti-FLAG antibodies, followed by Western blotting using antibody against TRIM33 and FLAG. D, PAR-bound ALC1 does not bind TRIM33 in vitro. WtALC1, KR (ATPase dead) and DA (PAR binding mutant) ALC1 mutants and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with PAR, washed, and then incubated with purified TRIM33. Membranes were then processed for Western blot analysis with antibodies to PAR (top panel) or antibody to TRIM33 (bottom panel).

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Western Blot, Immunoprecipitation, Quantitation Assay, Irradiation, In Vitro, Binding Assay, Mutagenesis, Membrane, Incubation, Purification

FIGURE 6. TRIM33 knockdown induces delayed dissociation of ALC1 but does not affect PAR dissociation from sites of DNA damage. A, HeLa cells expressingeithercontrolshRNA,TRIM33shRNA,TRIM33shRNAWtTRIM33,ortheTRIM33shRNATRIM33CAmutantweresubjectedtoUVlaserscissorsand stainedforH2AX(green)andALC1(red)atthetimepointsindicated.B,WesternblotanalysisshowinglevelsofTRIM33incellstreatedwithcontrolshRNA(lane 1), TRIM33 shRNA (lane 2), TRIM33sh RNA WtTRIM33 (lane 3), and TRIM33sh RNA TRIM33CA (lane 4). C, quantification of ALC1 localization to DNA damage in TRIM33 knockdown, WtTRIM33, or TRIM33CA reconstituted cells. Each data point is mean S.D. of at least 20 cells. D, TRIM33 knockdown does not affect ALC1 protein levels. Shown is a Western blot analysis showing levels of ALC1, TRIM33, and tubulin in control sh (1), TRIM33sh and TRIM33sh (2) cells reconstituted with either TRIM33Wt (3) or TRIM33CA (4). Protein extracts were collected from untreated or UV-treated cells, as indicated, and probed by Western blot analysis using the antibodies indicated. E, HeLa cells expressing either control shRNA or TRIM33 sh2 RNA were treated with laser scissors and stained by IF for H2AX (green) and PAR (red). F, quantification of PAR intensity was performed 5 and 45 min after induction of DNA damage. Data are plotted as the mean S.D. of at least 20 cells. *, p 0.005 for control versus TRIM33 shRNA-treated cells. G, Western blot analysis showing TRIM33 knockdown. HeLa cells transfected with control or TRIM33 shRNA and proteins levels were detected by Western blot analysis by probing with antibodies against TRIM33 and tubulin.

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE 6. TRIM33 knockdown induces delayed dissociation of ALC1 but does not affect PAR dissociation from sites of DNA damage. A, HeLa cells expressingeithercontrolshRNA,TRIM33shRNA,TRIM33shRNAWtTRIM33,ortheTRIM33shRNATRIM33CAmutantweresubjectedtoUVlaserscissorsand stainedforH2AX(green)andALC1(red)atthetimepointsindicated.B,WesternblotanalysisshowinglevelsofTRIM33incellstreatedwithcontrolshRNA(lane 1), TRIM33 shRNA (lane 2), TRIM33sh RNA WtTRIM33 (lane 3), and TRIM33sh RNA TRIM33CA (lane 4). C, quantification of ALC1 localization to DNA damage in TRIM33 knockdown, WtTRIM33, or TRIM33CA reconstituted cells. Each data point is mean S.D. of at least 20 cells. D, TRIM33 knockdown does not affect ALC1 protein levels. Shown is a Western blot analysis showing levels of ALC1, TRIM33, and tubulin in control sh (1), TRIM33sh and TRIM33sh (2) cells reconstituted with either TRIM33Wt (3) or TRIM33CA (4). Protein extracts were collected from untreated or UV-treated cells, as indicated, and probed by Western blot analysis using the antibodies indicated. E, HeLa cells expressing either control shRNA or TRIM33 sh2 RNA were treated with laser scissors and stained by IF for H2AX (green) and PAR (red). F, quantification of PAR intensity was performed 5 and 45 min after induction of DNA damage. Data are plotted as the mean S.D. of at least 20 cells. *, p 0.005 for control versus TRIM33 shRNA-treated cells. G, Western blot analysis showing TRIM33 knockdown. HeLa cells transfected with control or TRIM33 shRNA and proteins levels were detected by Western blot analysis by probing with antibodies against TRIM33 and tubulin.

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Knockdown, shRNA, Western Blot, Control, Expressing, Staining, Transfection

FIGURE 7. TRIM33 rescues ALC1 overexpression sensitivity to DNA-damaging agents. A, ALC1 overexpression results in delayed dissociation of ALC1 from DNAdamage.FLP-InALC1andFLP-InALC1WtTRIM33cellswereexposedtoUVlaserscissor-inducedDNAdamage,andcellswerefixedattheindicatedtime points. Cells were stained by IF for H2AX (green) and ALC1 (red). B, Western blot analysis showing levels of TRIM33 and ALC1 in ALC1-overexpressing cells. C, quantification of ALC1 intensity was performed at the indicated time points after induction of DNA damage. Data are plotted as the mean S.D. of at least 20 cells. D, effect of TRIM33 on sensitivity of ALC1-overexpressing cells to bleomycin. U2OS cells stably expressing either empty vector (FLP-In-FLAG), WtALC1 (FLP-In-ALC1),WtALC1(FLP-In-ALC1)WtTRIM33,andWtALC1(FLP-In-ALC1)TRIM33CAweretreatedwithincreasingconcentrationsofbleomycinfor3days. Relative cell counts at day 3 were measured by MTS assay and were plotted normalized to untreated cells. Data are mean S.D. of three experiments. E, induction of DNA breaks in cells (n 200 cells) of indicated genotypes after treatment with increasing amounts of phleomycin were measured by alkaline comet assay. Data are plotted as the mean S.D. of at least three experiments.

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m113.459164

Figure Lengend Snippet: FIGURE 7. TRIM33 rescues ALC1 overexpression sensitivity to DNA-damaging agents. A, ALC1 overexpression results in delayed dissociation of ALC1 from DNAdamage.FLP-InALC1andFLP-InALC1WtTRIM33cellswereexposedtoUVlaserscissor-inducedDNAdamage,andcellswerefixedattheindicatedtime points. Cells were stained by IF for H2AX (green) and ALC1 (red). B, Western blot analysis showing levels of TRIM33 and ALC1 in ALC1-overexpressing cells. C, quantification of ALC1 intensity was performed at the indicated time points after induction of DNA damage. Data are plotted as the mean S.D. of at least 20 cells. D, effect of TRIM33 on sensitivity of ALC1-overexpressing cells to bleomycin. U2OS cells stably expressing either empty vector (FLP-In-FLAG), WtALC1 (FLP-In-ALC1),WtALC1(FLP-In-ALC1)WtTRIM33,andWtALC1(FLP-In-ALC1)TRIM33CAweretreatedwithincreasingconcentrationsofbleomycinfor3days. Relative cell counts at day 3 were measured by MTS assay and were plotted normalized to untreated cells. Data are mean S.D. of three experiments. E, induction of DNA breaks in cells (n 200 cells) of indicated genotypes after treatment with increasing amounts of phleomycin were measured by alkaline comet assay. Data are plotted as the mean S.D. of at least three experiments.

Article Snippet: GST-tagged WtTRIM33 (plasmid 15734, Addgene), TRIM33 ShRNA (plasmid 15728, Addgene), and TRIM33 GFP shRNA (plasmid 15721, Addgene) were as described previously (26).

Techniques: Over Expression, Staining, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, MTS Assay, Alkaline Single Cell Gel Electrophoresis

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1) Product Images from "KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5."

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