Review



rabbit polyclonal antibody against trim39  (Proteintech)


Bioz Verified Symbol Proteintech is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 91
      Buy from Supplier

    Structured Review

    Proteintech rabbit polyclonal antibody against trim39
    A. BHK cells were transfected with Trim17-GFP together with <t>Flag-Trim39</t> or empty plasmid (as a negative control) for 24 h. Cells were then treated with 20 μM MG-132 for 5 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-Flag agarose beads (left panel) or GFP-Trap beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-GFP and anti-Flag antibodies. B. Neuro2A cells were transfected with HA-NFATc3 together with Flag-Trim39 or empty plasmid for 24 h. Cells were then treated as in A and lysates were subjected to immunoprecipitation using anti-Flag (left panel) or anti-HA (right panel) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. C. Neuro2A cells were treated with 10 μM MG-132 for 4 h and then fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Negative control was obtained by omitting anti-Trim39 antibody. When indicated, cells were previously transfected with Trim39 for 24 h (overexpressed). Images were analyzed by confocal microscopy. To better visualize the differences in PLA intensity, maximum intensity projection was applied to the z-stacks of images on the left panel. To better determine the subcellular location of the NFATc3/Trim39 interaction, a single slice of the z-stack is presented on the right panel (endo 1 slice). Nuclear staining was performed using DAPI. D. Neuro2A cells were transduced with lentiviral particles expressing a control shRNA (sheGFP) or a specific shRNA against Trim39 (shTrim39#1) for 24 h. Transduced cells were selected using puromycin for two additional days and plated onto coverslips. The day after plating, cells were analyzed by immunofluorescence using two different antibodies against Trim39; in red: antibody from Origene, in green: antibody from Proteintech. Images were set to the same minimum and maximum intensity to allow signal intensity comparison. E. Additional coverslips from the experiment presented in D were treated as in C and z-stacks of images were subjected to maximum intensity projection.
    Rabbit Polyclonal Antibody Against Trim39, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal antibody against trim39/product/Proteintech
    Average 91 stars, based on 1 article reviews
    rabbit polyclonal antibody against trim39 - by Bioz Stars, 2026-05
    91/100 stars

    Images

    1) Product Images from "Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3"

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    Journal: bioRxiv

    doi: 10.1101/2020.09.29.317958

    A. BHK cells were transfected with Trim17-GFP together with Flag-Trim39 or empty plasmid (as a negative control) for 24 h. Cells were then treated with 20 μM MG-132 for 5 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-Flag agarose beads (left panel) or GFP-Trap beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-GFP and anti-Flag antibodies. B. Neuro2A cells were transfected with HA-NFATc3 together with Flag-Trim39 or empty plasmid for 24 h. Cells were then treated as in A and lysates were subjected to immunoprecipitation using anti-Flag (left panel) or anti-HA (right panel) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. C. Neuro2A cells were treated with 10 μM MG-132 for 4 h and then fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Negative control was obtained by omitting anti-Trim39 antibody. When indicated, cells were previously transfected with Trim39 for 24 h (overexpressed). Images were analyzed by confocal microscopy. To better visualize the differences in PLA intensity, maximum intensity projection was applied to the z-stacks of images on the left panel. To better determine the subcellular location of the NFATc3/Trim39 interaction, a single slice of the z-stack is presented on the right panel (endo 1 slice). Nuclear staining was performed using DAPI. D. Neuro2A cells were transduced with lentiviral particles expressing a control shRNA (sheGFP) or a specific shRNA against Trim39 (shTrim39#1) for 24 h. Transduced cells were selected using puromycin for two additional days and plated onto coverslips. The day after plating, cells were analyzed by immunofluorescence using two different antibodies against Trim39; in red: antibody from Origene, in green: antibody from Proteintech. Images were set to the same minimum and maximum intensity to allow signal intensity comparison. E. Additional coverslips from the experiment presented in D were treated as in C and z-stacks of images were subjected to maximum intensity projection.
    Figure Legend Snippet: A. BHK cells were transfected with Trim17-GFP together with Flag-Trim39 or empty plasmid (as a negative control) for 24 h. Cells were then treated with 20 μM MG-132 for 5 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-Flag agarose beads (left panel) or GFP-Trap beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-GFP and anti-Flag antibodies. B. Neuro2A cells were transfected with HA-NFATc3 together with Flag-Trim39 or empty plasmid for 24 h. Cells were then treated as in A and lysates were subjected to immunoprecipitation using anti-Flag (left panel) or anti-HA (right panel) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. C. Neuro2A cells were treated with 10 μM MG-132 for 4 h and then fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Negative control was obtained by omitting anti-Trim39 antibody. When indicated, cells were previously transfected with Trim39 for 24 h (overexpressed). Images were analyzed by confocal microscopy. To better visualize the differences in PLA intensity, maximum intensity projection was applied to the z-stacks of images on the left panel. To better determine the subcellular location of the NFATc3/Trim39 interaction, a single slice of the z-stack is presented on the right panel (endo 1 slice). Nuclear staining was performed using DAPI. D. Neuro2A cells were transduced with lentiviral particles expressing a control shRNA (sheGFP) or a specific shRNA against Trim39 (shTrim39#1) for 24 h. Transduced cells were selected using puromycin for two additional days and plated onto coverslips. The day after plating, cells were analyzed by immunofluorescence using two different antibodies against Trim39; in red: antibody from Origene, in green: antibody from Proteintech. Images were set to the same minimum and maximum intensity to allow signal intensity comparison. E. Additional coverslips from the experiment presented in D were treated as in C and z-stacks of images were subjected to maximum intensity projection.

    Techniques Used: Transfection, Plasmid Preparation, Negative Control, Immunoprecipitation, Western Blot, In Situ, Confocal Microscopy, Staining, Transduction, Expressing, Control, shRNA, Immunofluorescence, Comparison

    A. Neuro2A cells were transfected with HA-NFATc3 together with empty plasmid or His-tagged ubiquitin, in the presence or absence of Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Cells were then incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transduced with lentiviruses expressing a control shRNA (directed against eGFP) or three different shRNAs targeting Trim39. Following 24 h transduction and 48 h selection of transduced cells using puromycin, cells were plated in new dishes. Then, cells were transfected with HA-NFATc3 or His-tagged ubiquitin or both for 24 h, and treated as in A. In conditions indicated by a star (*), some material was lost during TCA precipitation of the input lysate without affecting the amount of proteins in the nickel bead purification. These data are representative of 4 independent experiments. C. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing E1 and E2 enzymes) with purified recombinant GST-Trim39 (WT) or its inactive mutant GST-Trim39-C49S/C52S (mut) in the presence or the absence of ubiquitin as indicated. Poly-ubiquitinated forms of NFATc3 were detected by immunoblotting using an anti-NFATc3 antibody. The same membrane was immunoblotted with an anti-TRIM39 antibody to verify that similar amounts of recombinant WT GST-Trim39 and GST-Trim39-C49S/C52S were used in the assay. Note that in the presence of ubiquitin the unmodified form of WT GST-Trim39 is lower due to high Trim39 ubiquitination.
    Figure Legend Snippet: A. Neuro2A cells were transfected with HA-NFATc3 together with empty plasmid or His-tagged ubiquitin, in the presence or absence of Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Cells were then incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transduced with lentiviruses expressing a control shRNA (directed against eGFP) or three different shRNAs targeting Trim39. Following 24 h transduction and 48 h selection of transduced cells using puromycin, cells were plated in new dishes. Then, cells were transfected with HA-NFATc3 or His-tagged ubiquitin or both for 24 h, and treated as in A. In conditions indicated by a star (*), some material was lost during TCA precipitation of the input lysate without affecting the amount of proteins in the nickel bead purification. These data are representative of 4 independent experiments. C. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing E1 and E2 enzymes) with purified recombinant GST-Trim39 (WT) or its inactive mutant GST-Trim39-C49S/C52S (mut) in the presence or the absence of ubiquitin as indicated. Poly-ubiquitinated forms of NFATc3 were detected by immunoblotting using an anti-NFATc3 antibody. The same membrane was immunoblotted with an anti-TRIM39 antibody to verify that similar amounts of recombinant WT GST-Trim39 and GST-Trim39-C49S/C52S were used in the assay. Note that in the presence of ubiquitin the unmodified form of WT GST-Trim39 is lower due to high Trim39 ubiquitination.

    Techniques Used: Transfection, Plasmid Preparation, Ubiquitin Proteomics, Mutagenesis, Incubation, Purification, Western Blot, SDS Page, Transduction, Expressing, Control, shRNA, Selection, TCA Precipitation, In Vitro, Immu-Puri, Recombinant, Membrane

    A. BHK cells were transfected with a fixed amount of a HA-NFATc3 expressing vector (1 μg) together with empty plasmid (−) or increasing amounts of Flag-Trim39 expressing vector (0.1, 0.2, 0.5 and 1 μg) or 0.2 μg of a vector expressing the inactive mutant Flag-Trim39-ΔRING. When indicated, the cells were treated with 10 μM MG-132 for 6 h before harvesting. Total lysates were analyzed by western blot using antibodies against HA, Flag and actin. B. Neuro2A cells were left untreated (NT), or transfected twice with an siRNA targeting specifically Trim39 (siTrim39#1) or with a negative control siRNA (siCtrl) for 48 h. Total lysates were analyzed by western blot using antibodies against NFATc3, Trim39 and actin. The intensity of the NFATc3 bands on the western blots was quantified, normalized by the intensity of the actin bands and expressed relative to the values obtained with the control shRNA. The graph shows mean ± SEM from three independent experiments. **P<0.01 significantly different from siCtrl (one-way ANOVA followed by Dunnett’s multiple comparisons test). C. Neuro2A cells were co-transfected with empty plasmid or HA-NFATc3, together with empty plasmid, Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Then, total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of four independent experiments. **P<0.01 significantly different from the corresponding control (two-way ANOVA followed by Sidak’s multiple comparisons test). D. Neuro2A cells were transfected with empty plasmid, Flag-Trim39 or Flag-Trim39-ΔRING for 24 h. Then, cells were left untreated (control) or were deprived of serum for 3 h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 1 h (A23+PMA). Total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of three independent experiments. *P<0.05; **P<0.01 significantly different from the corresponding value in cells transfected with empty plasmid (two-way ANOVA followed by Sidak’s multiple comparisons test). E. Neuro2A cells were transfected twice with two different siRNAs targeting specifically Trim39 or with a negative control siRNA for 48 h. Then, cells were left untreated (control) or were deprived of serum for 3h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 30 min (A23+PMA). Total RNA was extracted and the mRNA level of Trim39 (left panel) or Trim17 (right panel) was estimated by quantitative PCR (NT = non transfected). Data are the means ± SEM of six independent experiments. *P<0.05; ***P<0.001 significantly different from cells transfected with control siRNA in the same condition (two-way ANOVA followed by Sidak’s multiple comparisons test).
    Figure Legend Snippet: A. BHK cells were transfected with a fixed amount of a HA-NFATc3 expressing vector (1 μg) together with empty plasmid (−) or increasing amounts of Flag-Trim39 expressing vector (0.1, 0.2, 0.5 and 1 μg) or 0.2 μg of a vector expressing the inactive mutant Flag-Trim39-ΔRING. When indicated, the cells were treated with 10 μM MG-132 for 6 h before harvesting. Total lysates were analyzed by western blot using antibodies against HA, Flag and actin. B. Neuro2A cells were left untreated (NT), or transfected twice with an siRNA targeting specifically Trim39 (siTrim39#1) or with a negative control siRNA (siCtrl) for 48 h. Total lysates were analyzed by western blot using antibodies against NFATc3, Trim39 and actin. The intensity of the NFATc3 bands on the western blots was quantified, normalized by the intensity of the actin bands and expressed relative to the values obtained with the control shRNA. The graph shows mean ± SEM from three independent experiments. **P<0.01 significantly different from siCtrl (one-way ANOVA followed by Dunnett’s multiple comparisons test). C. Neuro2A cells were co-transfected with empty plasmid or HA-NFATc3, together with empty plasmid, Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Then, total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of four independent experiments. **P<0.01 significantly different from the corresponding control (two-way ANOVA followed by Sidak’s multiple comparisons test). D. Neuro2A cells were transfected with empty plasmid, Flag-Trim39 or Flag-Trim39-ΔRING for 24 h. Then, cells were left untreated (control) or were deprived of serum for 3 h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 1 h (A23+PMA). Total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of three independent experiments. *P<0.05; **P<0.01 significantly different from the corresponding value in cells transfected with empty plasmid (two-way ANOVA followed by Sidak’s multiple comparisons test). E. Neuro2A cells were transfected twice with two different siRNAs targeting specifically Trim39 or with a negative control siRNA for 48 h. Then, cells were left untreated (control) or were deprived of serum for 3h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 30 min (A23+PMA). Total RNA was extracted and the mRNA level of Trim39 (left panel) or Trim17 (right panel) was estimated by quantitative PCR (NT = non transfected). Data are the means ± SEM of six independent experiments. *P<0.05; ***P<0.001 significantly different from cells transfected with control siRNA in the same condition (two-way ANOVA followed by Sidak’s multiple comparisons test).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Negative Control, Control, shRNA, Real-time Polymerase Chain Reaction

    A. BHK cells were transfected with HA-NFATc3 together with His-tagged ubiquitin, in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA and anti-Flag antibodies to detect poly-ubiquitinated forms of NFATc3 and Trim39. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA, Flag and GFP. B. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing ubiquitin and E1 and E2 enzymes) with purified recombinant His-TRIM39 or MBP-TRIM17 as indicated. Poly-ubiquitinated forms of NFATc3, TRIM39 and TRIM17 were detected by immunoblotting using anti-NFATc3, anti-TRIM39 and anti-TRIM17 antibodies revealed using high exposure times. Low exposure times were used to compare the level of TRIM39 and TRIM17 in the different conditions.
    Figure Legend Snippet: A. BHK cells were transfected with HA-NFATc3 together with His-tagged ubiquitin, in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA and anti-Flag antibodies to detect poly-ubiquitinated forms of NFATc3 and Trim39. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA, Flag and GFP. B. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing ubiquitin and E1 and E2 enzymes) with purified recombinant His-TRIM39 or MBP-TRIM17 as indicated. Poly-ubiquitinated forms of NFATc3, TRIM39 and TRIM17 were detected by immunoblotting using anti-NFATc3, anti-TRIM39 and anti-TRIM17 antibodies revealed using high exposure times. Low exposure times were used to compare the level of TRIM39 and TRIM17 in the different conditions.

    Techniques Used: Transfection, Ubiquitin Proteomics, Incubation, Purification, Western Blot, SDS Page, In Vitro, Immu-Puri, Recombinant

    A,B. Neuro2A cells were transfected with HA-NFATc3 in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Cells were then treated with 20 μM MG-132 for 7 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA (A) or anti-Flag (B) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA, anti-GFP and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands corresponding to immunoprecipitated HA-NFATc3 (A). The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands corresponding to immunoprecipitated Flag-Trim39 (B). Relative values are indicated in red. C. Neuro2A cells were transfected with GFP or Trim17-GFP for 24 h. Then cells were treated with 10 μM MG-132 for 4 h, fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Images were analyzed by confocal microscopy and a single slice of the z-stacks is presented for each condition. Nuclear staining was performed using DAPI. Note that, in the Trim17-GFP condition, transfected cells (delineated by a yellow line) show less dots than neighboring non transfected cells, which is not the case in the GFP condition. D. The number of dots was determined in individual cells transfected with either GFP or Trim17-GFP using Fiji. Data represent one experiment, including 68 transfected cells for each condition, representative of two independent experiments. ****p<0.0001, significantly different from GFP transfected cells (unpaired t test).
    Figure Legend Snippet: A,B. Neuro2A cells were transfected with HA-NFATc3 in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Cells were then treated with 20 μM MG-132 for 7 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA (A) or anti-Flag (B) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA, anti-GFP and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands corresponding to immunoprecipitated HA-NFATc3 (A). The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands corresponding to immunoprecipitated Flag-Trim39 (B). Relative values are indicated in red. C. Neuro2A cells were transfected with GFP or Trim17-GFP for 24 h. Then cells were treated with 10 μM MG-132 for 4 h, fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Images were analyzed by confocal microscopy and a single slice of the z-stacks is presented for each condition. Nuclear staining was performed using DAPI. Note that, in the Trim17-GFP condition, transfected cells (delineated by a yellow line) show less dots than neighboring non transfected cells, which is not the case in the GFP condition. D. The number of dots was determined in individual cells transfected with either GFP or Trim17-GFP using Fiji. Data represent one experiment, including 68 transfected cells for each condition, representative of two independent experiments. ****p<0.0001, significantly different from GFP transfected cells (unpaired t test).

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, In Situ, Confocal Microscopy, Staining

    A. Neuro2A cells were transfected with His-tagged ubiquitin together with WT HA-NFATc3 or HA-NFATc3 EallA, in the presence or the absence of Flag-Trim39, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transfected with Flag-Trim39 together with WT HA-NFATc3, HA-NFATc3-EallA or empty plasmid for 24 h. Cells were then treated with 10 μM MG-132 for 8 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA antibody (left panel) or anti-Flag beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands of immunoprecipitated Flag-Trim39. Relative values are indicated in red. C. Recombinant GST, GST-Trim39 and its different SIM mutants were purified using glutathione beads and subsequently incubated with purified recombinant SUMO-2 and SUMO-2 chains. Material bound to the beads was eluted and analyzed by western blot using anti-SUMO and anti-GST antibodies. A small fraction of the SUMO-2 chains was also loaded on the gel (input) for comparison. The intensity of bound SUMO-chain bands was quantified and normalized by the intensity of corresponding GST-Trim39 bands. Relative values are indicated in red. Note that SUMO bands are multiple of ≈ 15 kDa corresponding to mono-, di-, tri-, tetra-SUMO etc… D. Neuro2A cells were transfected with HA-NFATc3 or empty plasmid together with WT Flag-Trim39 or its SIM3 mutant for 24 h. Cells were treated as in B and lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoprecipitates and total lysates were analyzed as in B. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. Relative values are indicated in red. E. Neuro2A cells were transfected with His-tagged ubiquitin (or empty plasmid) together with HA-NFATc3 in the presence or the absence of Flag-Trim39 or its SIM3 mutant, for 24 h. Then, cells were treated as in A. Ubiquitinated proteins and input lysates were analyzed as in A. The intensity of the ubiquitinated forms of NFATc3 was quantified and normalized by the intensity of NFATc3 in the total lysate. Relative values are indicated in red.
    Figure Legend Snippet: A. Neuro2A cells were transfected with His-tagged ubiquitin together with WT HA-NFATc3 or HA-NFATc3 EallA, in the presence or the absence of Flag-Trim39, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transfected with Flag-Trim39 together with WT HA-NFATc3, HA-NFATc3-EallA or empty plasmid for 24 h. Cells were then treated with 10 μM MG-132 for 8 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA antibody (left panel) or anti-Flag beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands of immunoprecipitated Flag-Trim39. Relative values are indicated in red. C. Recombinant GST, GST-Trim39 and its different SIM mutants were purified using glutathione beads and subsequently incubated with purified recombinant SUMO-2 and SUMO-2 chains. Material bound to the beads was eluted and analyzed by western blot using anti-SUMO and anti-GST antibodies. A small fraction of the SUMO-2 chains was also loaded on the gel (input) for comparison. The intensity of bound SUMO-chain bands was quantified and normalized by the intensity of corresponding GST-Trim39 bands. Relative values are indicated in red. Note that SUMO bands are multiple of ≈ 15 kDa corresponding to mono-, di-, tri-, tetra-SUMO etc… D. Neuro2A cells were transfected with HA-NFATc3 or empty plasmid together with WT Flag-Trim39 or its SIM3 mutant for 24 h. Cells were treated as in B and lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoprecipitates and total lysates were analyzed as in B. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. Relative values are indicated in red. E. Neuro2A cells were transfected with His-tagged ubiquitin (or empty plasmid) together with HA-NFATc3 in the presence or the absence of Flag-Trim39 or its SIM3 mutant, for 24 h. Then, cells were treated as in A. Ubiquitinated proteins and input lysates were analyzed as in A. The intensity of the ubiquitinated forms of NFATc3 was quantified and normalized by the intensity of NFATc3 in the total lysate. Relative values are indicated in red.

    Techniques Used: Transfection, Ubiquitin Proteomics, Incubation, Purification, Western Blot, SDS Page, Plasmid Preparation, Immunoprecipitation, Recombinant, Comparison, Mutagenesis

    A. CGN primary cultures were transfected after 5 days in vitro (DIV 5) with GFP (as a negative control), WT GFP-NFATc3 or GFP-NFATc3-EallA for 16 h. Then, neurons were switched to serum-free medium containing 5 mM KCl (K5) for 7 h or were left untreated (control). Following fixation, nuclei were visualized by DAPI staining and proteins fused to GFP were detected by fluorescent microscopy. Arrows indicate GFP-positive neurons with thick arrows for neurons undergoing apoptosis and thin arrows for healthy neurons. B. The percentage of transfected, GFP-positive neurons undergoing apoptosis was assessed by examining cell morphology and nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in A. * P <0.05; *** * P <0.001 significantly different from the corresponding value obtained in neurons transfected with GFP (two-way ANOVA followed by Sidak’s multiple comparisons test). C. CGNs were transduced with lentiviral particles expressing a non-targeting control (directed against Luciferase) or an shRNA specifically targeting Trim39 one day after plating. At DIV 6, total cell extracts from KCl-deprived neurons were analyzed by western blot using anti-Trim39 antibody (Origene). D. CGN were transduced and treated as in C. At DIV 6 they were incubated for 8 h in K5 medium, fixed and stained with DAPI. E. The percentage of apoptotic neurons was estimated by examining nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in D. **** P <0.0001 significantly different from neurons transduced with the control shRNA (two-way ANOVA followed by Sidak’s multiple comparisons test).
    Figure Legend Snippet: A. CGN primary cultures were transfected after 5 days in vitro (DIV 5) with GFP (as a negative control), WT GFP-NFATc3 or GFP-NFATc3-EallA for 16 h. Then, neurons were switched to serum-free medium containing 5 mM KCl (K5) for 7 h or were left untreated (control). Following fixation, nuclei were visualized by DAPI staining and proteins fused to GFP were detected by fluorescent microscopy. Arrows indicate GFP-positive neurons with thick arrows for neurons undergoing apoptosis and thin arrows for healthy neurons. B. The percentage of transfected, GFP-positive neurons undergoing apoptosis was assessed by examining cell morphology and nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in A. * P <0.05; *** * P <0.001 significantly different from the corresponding value obtained in neurons transfected with GFP (two-way ANOVA followed by Sidak’s multiple comparisons test). C. CGNs were transduced with lentiviral particles expressing a non-targeting control (directed against Luciferase) or an shRNA specifically targeting Trim39 one day after plating. At DIV 6, total cell extracts from KCl-deprived neurons were analyzed by western blot using anti-Trim39 antibody (Origene). D. CGN were transduced and treated as in C. At DIV 6 they were incubated for 8 h in K5 medium, fixed and stained with DAPI. E. The percentage of apoptotic neurons was estimated by examining nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in D. **** P <0.0001 significantly different from neurons transduced with the control shRNA (two-way ANOVA followed by Sidak’s multiple comparisons test).

    Techniques Used: Transfection, In Vitro, Negative Control, Control, Staining, Microscopy, Transduction, Expressing, Luciferase, shRNA, Western Blot, Incubation



    Similar Products

    rabbit polyclonal antibody against trim39  (Proteintech)
    91
    Proteintech rabbit polyclonal antibody against trim39
    A. BHK cells were transfected with Trim17-GFP together with <t>Flag-Trim39</t> or empty plasmid (as a negative control) for 24 h. Cells were then treated with 20 μM MG-132 for 5 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-Flag agarose beads (left panel) or GFP-Trap beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-GFP and anti-Flag antibodies. B. Neuro2A cells were transfected with HA-NFATc3 together with Flag-Trim39 or empty plasmid for 24 h. Cells were then treated as in A and lysates were subjected to immunoprecipitation using anti-Flag (left panel) or anti-HA (right panel) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. C. Neuro2A cells were treated with 10 μM MG-132 for 4 h and then fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Negative control was obtained by omitting anti-Trim39 antibody. When indicated, cells were previously transfected with Trim39 for 24 h (overexpressed). Images were analyzed by confocal microscopy. To better visualize the differences in PLA intensity, maximum intensity projection was applied to the z-stacks of images on the left panel. To better determine the subcellular location of the NFATc3/Trim39 interaction, a single slice of the z-stack is presented on the right panel (endo 1 slice). Nuclear staining was performed using DAPI. D. Neuro2A cells were transduced with lentiviral particles expressing a control shRNA (sheGFP) or a specific shRNA against Trim39 (shTrim39#1) for 24 h. Transduced cells were selected using puromycin for two additional days and plated onto coverslips. The day after plating, cells were analyzed by immunofluorescence using two different antibodies against Trim39; in red: antibody from Origene, in green: antibody from Proteintech. Images were set to the same minimum and maximum intensity to allow signal intensity comparison. E. Additional coverslips from the experiment presented in D were treated as in C and z-stacks of images were subjected to maximum intensity projection.
    Rabbit Polyclonal Antibody Against Trim39, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal antibody against trim39/product/Proteintech
    Average 91 stars, based on 1 article reviews
    rabbit polyclonal antibody against trim39 - by Bioz Stars, 2026-05
    91/100 stars
    		  Buy from Supplier

    Image Search Results


    A. BHK cells were transfected with Trim17-GFP together with Flag-Trim39 or empty plasmid (as a negative control) for 24 h. Cells were then treated with 20 μM MG-132 for 5 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-Flag agarose beads (left panel) or GFP-Trap beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-GFP and anti-Flag antibodies. B. Neuro2A cells were transfected with HA-NFATc3 together with Flag-Trim39 or empty plasmid for 24 h. Cells were then treated as in A and lysates were subjected to immunoprecipitation using anti-Flag (left panel) or anti-HA (right panel) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. C. Neuro2A cells were treated with 10 μM MG-132 for 4 h and then fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Negative control was obtained by omitting anti-Trim39 antibody. When indicated, cells were previously transfected with Trim39 for 24 h (overexpressed). Images were analyzed by confocal microscopy. To better visualize the differences in PLA intensity, maximum intensity projection was applied to the z-stacks of images on the left panel. To better determine the subcellular location of the NFATc3/Trim39 interaction, a single slice of the z-stack is presented on the right panel (endo 1 slice). Nuclear staining was performed using DAPI. D. Neuro2A cells were transduced with lentiviral particles expressing a control shRNA (sheGFP) or a specific shRNA against Trim39 (shTrim39#1) for 24 h. Transduced cells were selected using puromycin for two additional days and plated onto coverslips. The day after plating, cells were analyzed by immunofluorescence using two different antibodies against Trim39; in red: antibody from Origene, in green: antibody from Proteintech. Images were set to the same minimum and maximum intensity to allow signal intensity comparison. E. Additional coverslips from the experiment presented in D were treated as in C and z-stacks of images were subjected to maximum intensity projection.

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A. BHK cells were transfected with Trim17-GFP together with Flag-Trim39 or empty plasmid (as a negative control) for 24 h. Cells were then treated with 20 μM MG-132 for 5 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-Flag agarose beads (left panel) or GFP-Trap beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-GFP and anti-Flag antibodies. B. Neuro2A cells were transfected with HA-NFATc3 together with Flag-Trim39 or empty plasmid for 24 h. Cells were then treated as in A and lysates were subjected to immunoprecipitation using anti-Flag (left panel) or anti-HA (right panel) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. C. Neuro2A cells were treated with 10 μM MG-132 for 4 h and then fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Negative control was obtained by omitting anti-Trim39 antibody. When indicated, cells were previously transfected with Trim39 for 24 h (overexpressed). Images were analyzed by confocal microscopy. To better visualize the differences in PLA intensity, maximum intensity projection was applied to the z-stacks of images on the left panel. To better determine the subcellular location of the NFATc3/Trim39 interaction, a single slice of the z-stack is presented on the right panel (endo 1 slice). Nuclear staining was performed using DAPI. D. Neuro2A cells were transduced with lentiviral particles expressing a control shRNA (sheGFP) or a specific shRNA against Trim39 (shTrim39#1) for 24 h. Transduced cells were selected using puromycin for two additional days and plated onto coverslips. The day after plating, cells were analyzed by immunofluorescence using two different antibodies against Trim39; in red: antibody from Origene, in green: antibody from Proteintech. Images were set to the same minimum and maximum intensity to allow signal intensity comparison. E. Additional coverslips from the experiment presented in D were treated as in C and z-stacks of images were subjected to maximum intensity projection.

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, Plasmid Preparation, Negative Control, Immunoprecipitation, Western Blot, In Situ, Confocal Microscopy, Staining, Transduction, Expressing, Control, shRNA, Immunofluorescence, Comparison

    A. Neuro2A cells were transfected with HA-NFATc3 together with empty plasmid or His-tagged ubiquitin, in the presence or absence of Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Cells were then incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transduced with lentiviruses expressing a control shRNA (directed against eGFP) or three different shRNAs targeting Trim39. Following 24 h transduction and 48 h selection of transduced cells using puromycin, cells were plated in new dishes. Then, cells were transfected with HA-NFATc3 or His-tagged ubiquitin or both for 24 h, and treated as in A. In conditions indicated by a star (*), some material was lost during TCA precipitation of the input lysate without affecting the amount of proteins in the nickel bead purification. These data are representative of 4 independent experiments. C. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing E1 and E2 enzymes) with purified recombinant GST-Trim39 (WT) or its inactive mutant GST-Trim39-C49S/C52S (mut) in the presence or the absence of ubiquitin as indicated. Poly-ubiquitinated forms of NFATc3 were detected by immunoblotting using an anti-NFATc3 antibody. The same membrane was immunoblotted with an anti-TRIM39 antibody to verify that similar amounts of recombinant WT GST-Trim39 and GST-Trim39-C49S/C52S were used in the assay. Note that in the presence of ubiquitin the unmodified form of WT GST-Trim39 is lower due to high Trim39 ubiquitination.

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A. Neuro2A cells were transfected with HA-NFATc3 together with empty plasmid or His-tagged ubiquitin, in the presence or absence of Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Cells were then incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transduced with lentiviruses expressing a control shRNA (directed against eGFP) or three different shRNAs targeting Trim39. Following 24 h transduction and 48 h selection of transduced cells using puromycin, cells were plated in new dishes. Then, cells were transfected with HA-NFATc3 or His-tagged ubiquitin or both for 24 h, and treated as in A. In conditions indicated by a star (*), some material was lost during TCA precipitation of the input lysate without affecting the amount of proteins in the nickel bead purification. These data are representative of 4 independent experiments. C. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing E1 and E2 enzymes) with purified recombinant GST-Trim39 (WT) or its inactive mutant GST-Trim39-C49S/C52S (mut) in the presence or the absence of ubiquitin as indicated. Poly-ubiquitinated forms of NFATc3 were detected by immunoblotting using an anti-NFATc3 antibody. The same membrane was immunoblotted with an anti-TRIM39 antibody to verify that similar amounts of recombinant WT GST-Trim39 and GST-Trim39-C49S/C52S were used in the assay. Note that in the presence of ubiquitin the unmodified form of WT GST-Trim39 is lower due to high Trim39 ubiquitination.

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, Plasmid Preparation, Ubiquitin Proteomics, Mutagenesis, Incubation, Purification, Western Blot, SDS Page, Transduction, Expressing, Control, shRNA, Selection, TCA Precipitation, In Vitro, Immu-Puri, Recombinant, Membrane

    A. BHK cells were transfected with a fixed amount of a HA-NFATc3 expressing vector (1 μg) together with empty plasmid (−) or increasing amounts of Flag-Trim39 expressing vector (0.1, 0.2, 0.5 and 1 μg) or 0.2 μg of a vector expressing the inactive mutant Flag-Trim39-ΔRING. When indicated, the cells were treated with 10 μM MG-132 for 6 h before harvesting. Total lysates were analyzed by western blot using antibodies against HA, Flag and actin. B. Neuro2A cells were left untreated (NT), or transfected twice with an siRNA targeting specifically Trim39 (siTrim39#1) or with a negative control siRNA (siCtrl) for 48 h. Total lysates were analyzed by western blot using antibodies against NFATc3, Trim39 and actin. The intensity of the NFATc3 bands on the western blots was quantified, normalized by the intensity of the actin bands and expressed relative to the values obtained with the control shRNA. The graph shows mean ± SEM from three independent experiments. **P<0.01 significantly different from siCtrl (one-way ANOVA followed by Dunnett’s multiple comparisons test). C. Neuro2A cells were co-transfected with empty plasmid or HA-NFATc3, together with empty plasmid, Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Then, total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of four independent experiments. **P<0.01 significantly different from the corresponding control (two-way ANOVA followed by Sidak’s multiple comparisons test). D. Neuro2A cells were transfected with empty plasmid, Flag-Trim39 or Flag-Trim39-ΔRING for 24 h. Then, cells were left untreated (control) or were deprived of serum for 3 h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 1 h (A23+PMA). Total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of three independent experiments. *P<0.05; **P<0.01 significantly different from the corresponding value in cells transfected with empty plasmid (two-way ANOVA followed by Sidak’s multiple comparisons test). E. Neuro2A cells were transfected twice with two different siRNAs targeting specifically Trim39 or with a negative control siRNA for 48 h. Then, cells were left untreated (control) or were deprived of serum for 3h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 30 min (A23+PMA). Total RNA was extracted and the mRNA level of Trim39 (left panel) or Trim17 (right panel) was estimated by quantitative PCR (NT = non transfected). Data are the means ± SEM of six independent experiments. *P<0.05; ***P<0.001 significantly different from cells transfected with control siRNA in the same condition (two-way ANOVA followed by Sidak’s multiple comparisons test).

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A. BHK cells were transfected with a fixed amount of a HA-NFATc3 expressing vector (1 μg) together with empty plasmid (−) or increasing amounts of Flag-Trim39 expressing vector (0.1, 0.2, 0.5 and 1 μg) or 0.2 μg of a vector expressing the inactive mutant Flag-Trim39-ΔRING. When indicated, the cells were treated with 10 μM MG-132 for 6 h before harvesting. Total lysates were analyzed by western blot using antibodies against HA, Flag and actin. B. Neuro2A cells were left untreated (NT), or transfected twice with an siRNA targeting specifically Trim39 (siTrim39#1) or with a negative control siRNA (siCtrl) for 48 h. Total lysates were analyzed by western blot using antibodies against NFATc3, Trim39 and actin. The intensity of the NFATc3 bands on the western blots was quantified, normalized by the intensity of the actin bands and expressed relative to the values obtained with the control shRNA. The graph shows mean ± SEM from three independent experiments. **P<0.01 significantly different from siCtrl (one-way ANOVA followed by Dunnett’s multiple comparisons test). C. Neuro2A cells were co-transfected with empty plasmid or HA-NFATc3, together with empty plasmid, Flag-Trim39 or the inactive mutant Flag-Trim39-ΔRING for 24 h. Then, total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of four independent experiments. **P<0.01 significantly different from the corresponding control (two-way ANOVA followed by Sidak’s multiple comparisons test). D. Neuro2A cells were transfected with empty plasmid, Flag-Trim39 or Flag-Trim39-ΔRING for 24 h. Then, cells were left untreated (control) or were deprived of serum for 3 h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 1 h (A23+PMA). Total RNA was extracted and the mRNA level of Trim17 was estimated by quantitative PCR. Data are the means ± SEM of three independent experiments. *P<0.05; **P<0.01 significantly different from the corresponding value in cells transfected with empty plasmid (two-way ANOVA followed by Sidak’s multiple comparisons test). E. Neuro2A cells were transfected twice with two different siRNAs targeting specifically Trim39 or with a negative control siRNA for 48 h. Then, cells were left untreated (control) or were deprived of serum for 3h and subsequently treated with 1 μM A23187 and 100 nM PMA in serum-free medium for 30 min (A23+PMA). Total RNA was extracted and the mRNA level of Trim39 (left panel) or Trim17 (right panel) was estimated by quantitative PCR (NT = non transfected). Data are the means ± SEM of six independent experiments. *P<0.05; ***P<0.001 significantly different from cells transfected with control siRNA in the same condition (two-way ANOVA followed by Sidak’s multiple comparisons test).

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Negative Control, Control, shRNA, Real-time Polymerase Chain Reaction

    A. BHK cells were transfected with HA-NFATc3 together with His-tagged ubiquitin, in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA and anti-Flag antibodies to detect poly-ubiquitinated forms of NFATc3 and Trim39. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA, Flag and GFP. B. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing ubiquitin and E1 and E2 enzymes) with purified recombinant His-TRIM39 or MBP-TRIM17 as indicated. Poly-ubiquitinated forms of NFATc3, TRIM39 and TRIM17 were detected by immunoblotting using anti-NFATc3, anti-TRIM39 and anti-TRIM17 antibodies revealed using high exposure times. Low exposure times were used to compare the level of TRIM39 and TRIM17 in the different conditions.

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A. BHK cells were transfected with HA-NFATc3 together with His-tagged ubiquitin, in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA and anti-Flag antibodies to detect poly-ubiquitinated forms of NFATc3 and Trim39. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA, Flag and GFP. B. In vitro translated HA-NFATc3 was first immunopurified from wheat germ extract using anti-HA antibody. Then, beads used for immunopurification of NFATc3 were incubated for 1 h at 37°C in the in vitro ubiquitination reaction mix (containing ubiquitin and E1 and E2 enzymes) with purified recombinant His-TRIM39 or MBP-TRIM17 as indicated. Poly-ubiquitinated forms of NFATc3, TRIM39 and TRIM17 were detected by immunoblotting using anti-NFATc3, anti-TRIM39 and anti-TRIM17 antibodies revealed using high exposure times. Low exposure times were used to compare the level of TRIM39 and TRIM17 in the different conditions.

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, Ubiquitin Proteomics, Incubation, Purification, Western Blot, SDS Page, In Vitro, Immu-Puri, Recombinant

    A,B. Neuro2A cells were transfected with HA-NFATc3 in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Cells were then treated with 20 μM MG-132 for 7 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA (A) or anti-Flag (B) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA, anti-GFP and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands corresponding to immunoprecipitated HA-NFATc3 (A). The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands corresponding to immunoprecipitated Flag-Trim39 (B). Relative values are indicated in red. C. Neuro2A cells were transfected with GFP or Trim17-GFP for 24 h. Then cells were treated with 10 μM MG-132 for 4 h, fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Images were analyzed by confocal microscopy and a single slice of the z-stacks is presented for each condition. Nuclear staining was performed using DAPI. Note that, in the Trim17-GFP condition, transfected cells (delineated by a yellow line) show less dots than neighboring non transfected cells, which is not the case in the GFP condition. D. The number of dots was determined in individual cells transfected with either GFP or Trim17-GFP using Fiji. Data represent one experiment, including 68 transfected cells for each condition, representative of two independent experiments. ****p<0.0001, significantly different from GFP transfected cells (unpaired t test).

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A,B. Neuro2A cells were transfected with HA-NFATc3 in the presence or the absence of Flag-Trim39, Trim17-GFP or both, as indicated, for 24 h. Cells were then treated with 20 μM MG-132 for 7 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA (A) or anti-Flag (B) antibodies. Immunoprecipitates and total lysates were analyzed by western blot using anti-HA, anti-GFP and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands corresponding to immunoprecipitated HA-NFATc3 (A). The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands corresponding to immunoprecipitated Flag-Trim39 (B). Relative values are indicated in red. C. Neuro2A cells were transfected with GFP or Trim17-GFP for 24 h. Then cells were treated with 10 μM MG-132 for 4 h, fixed and subjected to in situ PLA using rabbit anti-NFATc3 and mouse anti-Trim39 antibodies. Each bright red spot indicates that the two proteins are in close proximity. Images were analyzed by confocal microscopy and a single slice of the z-stacks is presented for each condition. Nuclear staining was performed using DAPI. Note that, in the Trim17-GFP condition, transfected cells (delineated by a yellow line) show less dots than neighboring non transfected cells, which is not the case in the GFP condition. D. The number of dots was determined in individual cells transfected with either GFP or Trim17-GFP using Fiji. Data represent one experiment, including 68 transfected cells for each condition, representative of two independent experiments. ****p<0.0001, significantly different from GFP transfected cells (unpaired t test).

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, Immunoprecipitation, Western Blot, In Situ, Confocal Microscopy, Staining

    A. Neuro2A cells were transfected with His-tagged ubiquitin together with WT HA-NFATc3 or HA-NFATc3 EallA, in the presence or the absence of Flag-Trim39, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transfected with Flag-Trim39 together with WT HA-NFATc3, HA-NFATc3-EallA or empty plasmid for 24 h. Cells were then treated with 10 μM MG-132 for 8 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA antibody (left panel) or anti-Flag beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands of immunoprecipitated Flag-Trim39. Relative values are indicated in red. C. Recombinant GST, GST-Trim39 and its different SIM mutants were purified using glutathione beads and subsequently incubated with purified recombinant SUMO-2 and SUMO-2 chains. Material bound to the beads was eluted and analyzed by western blot using anti-SUMO and anti-GST antibodies. A small fraction of the SUMO-2 chains was also loaded on the gel (input) for comparison. The intensity of bound SUMO-chain bands was quantified and normalized by the intensity of corresponding GST-Trim39 bands. Relative values are indicated in red. Note that SUMO bands are multiple of ≈ 15 kDa corresponding to mono-, di-, tri-, tetra-SUMO etc… D. Neuro2A cells were transfected with HA-NFATc3 or empty plasmid together with WT Flag-Trim39 or its SIM3 mutant for 24 h. Cells were treated as in B and lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoprecipitates and total lysates were analyzed as in B. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. Relative values are indicated in red. E. Neuro2A cells were transfected with His-tagged ubiquitin (or empty plasmid) together with HA-NFATc3 in the presence or the absence of Flag-Trim39 or its SIM3 mutant, for 24 h. Then, cells were treated as in A. Ubiquitinated proteins and input lysates were analyzed as in A. The intensity of the ubiquitinated forms of NFATc3 was quantified and normalized by the intensity of NFATc3 in the total lysate. Relative values are indicated in red.

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A. Neuro2A cells were transfected with His-tagged ubiquitin together with WT HA-NFATc3 or HA-NFATc3 EallA, in the presence or the absence of Flag-Trim39, for 24 h. Then, cells were incubated with 20 μM MG-132 for 6 h before harvesting. The ubiquitinated proteins were purified using nickel beads and analyzed by western blotting using anti-HA antibody to detect ubiquitin-conjugated HA-NFATc3. In a separate SDS-PAGE, samples of the input lysates used for the purification were analyzed with antibodies against HA and Flag. B. Neuro2A cells were transfected with Flag-Trim39 together with WT HA-NFATc3, HA-NFATc3-EallA or empty plasmid for 24 h. Cells were then treated with 10 μM MG-132 for 8 h. The cells were subsequently harvested and lysates were subjected to immunoprecipitation using anti-HA antibody (left panel) or anti-Flag beads (right panel). Immunoprecipitates and total lysates were analyzed by western blot using anti-HA and anti-Flag antibodies. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. The intensity of the bands containing HA-NFATc3 co-immunoprecipitated with Flag-Trim39 was normalized by the intensity of the bands of immunoprecipitated Flag-Trim39. Relative values are indicated in red. C. Recombinant GST, GST-Trim39 and its different SIM mutants were purified using glutathione beads and subsequently incubated with purified recombinant SUMO-2 and SUMO-2 chains. Material bound to the beads was eluted and analyzed by western blot using anti-SUMO and anti-GST antibodies. A small fraction of the SUMO-2 chains was also loaded on the gel (input) for comparison. The intensity of bound SUMO-chain bands was quantified and normalized by the intensity of corresponding GST-Trim39 bands. Relative values are indicated in red. Note that SUMO bands are multiple of ≈ 15 kDa corresponding to mono-, di-, tri-, tetra-SUMO etc… D. Neuro2A cells were transfected with HA-NFATc3 or empty plasmid together with WT Flag-Trim39 or its SIM3 mutant for 24 h. Cells were treated as in B and lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoprecipitates and total lysates were analyzed as in B. The intensity of the bands containing Flag-Trim39 co-immunoprecipitated with HA-NFATc3 was normalized by the intensity of the bands of immunoprecipitated HA-NFATc3. Relative values are indicated in red. E. Neuro2A cells were transfected with His-tagged ubiquitin (or empty plasmid) together with HA-NFATc3 in the presence or the absence of Flag-Trim39 or its SIM3 mutant, for 24 h. Then, cells were treated as in A. Ubiquitinated proteins and input lysates were analyzed as in A. The intensity of the ubiquitinated forms of NFATc3 was quantified and normalized by the intensity of NFATc3 in the total lysate. Relative values are indicated in red.

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, Ubiquitin Proteomics, Incubation, Purification, Western Blot, SDS Page, Plasmid Preparation, Immunoprecipitation, Recombinant, Comparison, Mutagenesis

    A. CGN primary cultures were transfected after 5 days in vitro (DIV 5) with GFP (as a negative control), WT GFP-NFATc3 or GFP-NFATc3-EallA for 16 h. Then, neurons were switched to serum-free medium containing 5 mM KCl (K5) for 7 h or were left untreated (control). Following fixation, nuclei were visualized by DAPI staining and proteins fused to GFP were detected by fluorescent microscopy. Arrows indicate GFP-positive neurons with thick arrows for neurons undergoing apoptosis and thin arrows for healthy neurons. B. The percentage of transfected, GFP-positive neurons undergoing apoptosis was assessed by examining cell morphology and nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in A. * P <0.05; *** * P <0.001 significantly different from the corresponding value obtained in neurons transfected with GFP (two-way ANOVA followed by Sidak’s multiple comparisons test). C. CGNs were transduced with lentiviral particles expressing a non-targeting control (directed against Luciferase) or an shRNA specifically targeting Trim39 one day after plating. At DIV 6, total cell extracts from KCl-deprived neurons were analyzed by western blot using anti-Trim39 antibody (Origene). D. CGN were transduced and treated as in C. At DIV 6 they were incubated for 8 h in K5 medium, fixed and stained with DAPI. E. The percentage of apoptotic neurons was estimated by examining nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in D. **** P <0.0001 significantly different from neurons transduced with the control shRNA (two-way ANOVA followed by Sidak’s multiple comparisons test).

    Journal: bioRxiv

    Article Title: Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

    doi: 10.1101/2020.09.29.317958

    Figure Lengend Snippet: A. CGN primary cultures were transfected after 5 days in vitro (DIV 5) with GFP (as a negative control), WT GFP-NFATc3 or GFP-NFATc3-EallA for 16 h. Then, neurons were switched to serum-free medium containing 5 mM KCl (K5) for 7 h or were left untreated (control). Following fixation, nuclei were visualized by DAPI staining and proteins fused to GFP were detected by fluorescent microscopy. Arrows indicate GFP-positive neurons with thick arrows for neurons undergoing apoptosis and thin arrows for healthy neurons. B. The percentage of transfected, GFP-positive neurons undergoing apoptosis was assessed by examining cell morphology and nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in A. * P <0.05; *** * P <0.001 significantly different from the corresponding value obtained in neurons transfected with GFP (two-way ANOVA followed by Sidak’s multiple comparisons test). C. CGNs were transduced with lentiviral particles expressing a non-targeting control (directed against Luciferase) or an shRNA specifically targeting Trim39 one day after plating. At DIV 6, total cell extracts from KCl-deprived neurons were analyzed by western blot using anti-Trim39 antibody (Origene). D. CGN were transduced and treated as in C. At DIV 6 they were incubated for 8 h in K5 medium, fixed and stained with DAPI. E. The percentage of apoptotic neurons was estimated by examining nuclear condensation. Data are the means ± S.E.M. of four independent experiments performed as in D. **** P <0.0001 significantly different from neurons transduced with the control shRNA (two-way ANOVA followed by Sidak’s multiple comparisons test).

    Article Snippet: Rabbit polyclonal antibody against Trim39 was from Proteintech (#12757-1-AP).

    Techniques: Transfection, In Vitro, Negative Control, Control, Staining, Microscopy, Transduction, Expressing, Luciferase, shRNA, Western Blot, Incubation