37-1100

antibody from Invitrogen Antibodies

Targeting: UBE2N MGC8489, UBC13, UbcH-ben

Provider product page for 37-1100

Western blot

ELISA

Other assay

Antibody data

Antibody Data
Antigen structure
References [32]
Comments [0]
Validations
Western blot [1]
Other assay [19]

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Product number: 37-1100 - Provider product page
Provider: Invitrogen Antibodies
Product name: UBC13 Monoclonal Antibody (4E 11)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 4E 11
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

UBE2N protein structure - 37-1100 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 NSC697923 shows cytotoxic effect on NB cell lines. ( a ) A panel of six NB cell lines were lysed, subjected to SDS-PAGE and immunoblotted with UBE2N antibodies. Beta-actin was detected as a loading control. ( b ) Six NB cell lines were treated with the indicated concentrations of NSC697923 for 24 h. Cell viability was then measured by adding 10 mu l of CCK-8 and reading the absorbance at 450 nm. Data were represented as mean+-S.D. P -values

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 STAT3 suppresses osteoclastogenesis and RANK signal transduction (a) Femoral sections from Stat3 -deficient ( Stat3 delta/delta ) and -sufficient ( Stat3 +/+ ) mice, 8 weeks. ( Upper ) Hematoxylin and eosin (HE); ( Lower ) TRAP staining (arrows). Scale bar, 100 M. ( b to i ) Bone marrow cells from Stat3 +/+ and Stat3 delta/delta mice were cultured in M-CSF-containing medium for 4 d to generate bone marrow-derived macrophages. ( b ) 2x10 5 cells were cultured in M-CSF and RANKL (0, 25 or 50 ng mL -1 ) for an additional 4 d. Osteoclasts detected by TRAP staining (pink). Scale bar, 100 M ( c to f ) Macrophages were starved of exogenous growth factor overnight and stimulated with RANKL (100 ng mL -1 ) or left untreated, as indicated. ( c and d ) TRAF6 ubiquitination was evaluated -/+ RANKL stimulation by immunoprecipitation and immunoblotting for total ubiquitin ( c ) or K63-specific linkage ( d ). Lower panels: total TRAF6. ( e ) Nuclear NF-kappaB DNA binding activity was measured -/+ RANKL stimulation (15 min) by EMSA. Loading control: NF-Y EMSA. ( f ) MAPK activation was measured -/+ RANKL stimulation by immunoblotting whole cell lysates with antibodies specific for phosphorylated protein (pJNK, p-p38 or pERK). Total protein amounts served as loading controls (JNK, p38 or ERK). ( g ) Macrophages were cultured in M-CSF and RANKL (50 ng mL -1 ). TRAF6 and Ubc13 amounts were determined by immunoblotting. Loading control: RAN. The relative level of Ubc13 is expressed as a

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Autocrine IL-6 is required to repress Ubc13 expression a ) Il6 mRNA expression was measured in bone marrow-derived macrophages by qPCR following RANKL (50 ng mL -1 ) stimulation, as indicated. ( b and c ) Osteoclast gene expression (b) and Ubc13 amounts (c) were determined in bone marrow-derived macrophages following culture in M-CSF- and RANKL-containing medium without (-) or with (+) IL-6 blocking antibody by qPCR or immunoblotting, respectively. (d to f) Ubc13 amounts, nuclear NF-kappaB DNA binding activity and osteoclast gene expression were determined in Il6 +/+ and Il6 -/- bone marrow-derived macrophages following culture in M-CSF- and RANKL-containing medium for 1 or 2 d ( d ), 1 d ( e ), or 3 d ( f ) by immunoblotting, EMSA or qPCR, respectively. (g) Ubc13 expression was evaluated in bone marrow-derived macrophages from Stat3 -sufficient ( Stat3 +/+ ) or hematopoietic Stat3 -deficient ( Stat3 delta/delta ) mice by immunoblotting following IL-6 stimulation, as indicated. ( c , d and g ) Relative Ubc13 expression vs. untreated ( c ), IL-6-( d ) or STAT3-sufficient control ( g ) was calculated as indicated in Fig. 1g and displayed below bottom panels. ( a and f ) Results represent mean values of 3 independent experiments. Error bars indicate SEM. ( a and b ) Two-way Anova with Bonferroni multiple comparison used; ( f ) t test used. *, p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 STAT3 transcriptional activity is required to repress Ube2n expression ( a ) Ube2n mRNA was measured in bone marrow-derived macrophages from Stat3 -sufficient mice following treatment with IL-6 as indicated, using qPCR. (b and c) Ube2n promoter activity was measured by reporter assays in HEK293T cells transfected with pGL3- Ube2n ( Ube2n ), pGL3- Ube2n -mut ( Ube2n -mut; containing a mutated STATx site), pMX vector (vector), pMX-STAT3C (STAT3C), pMX-STAT3 DN (STAT3 DN) and pTK-Renilla, as indicated. pMX-STAT3 DN and pMX-STAT3C were included in some samples in a 3:1 or 6:1 ratio, as shown. (d) Stat3 -/- MEFs were transfected with pMX-STAT3C, pMX-STAT3 DN or both, at the ratios indicated. The expression level of pSTAT3, total STAT3 and Ubc13 was detected by immunoblotting. Ran served as loading control . (e and f) Bone marrow-derived macrophages from Stat3 -sufficient mice were cultured in M-CSF- and RANKL-containing medium in the absence or presence of the STAT3 inhibitor PM-73G, as indicated. Ubc13 amounts were detected by immunoblotting ( e ); osteoclast gene expression was measured by qPCR ( f ). ( d and e ) Relative Ubc13 expression vs. STAT3C transfection alone ( d ) or untreated cells on 1 d ( e ) was calculated as indicated in Fig. 1g and displayed below bottom panels. (a, b, c and f) Data represent mean values of 3 independent experiments. Error bars indicate SEM. Two-way Anova with Bonferroni multiple comparison used. *, p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Binding interface of MavC with Ube2N. a-c Detailed view of interactions in MavC regions 1, 2, and 3 with Ube2N. Key residues are labeled and represented as stick models. MavC is depicted in burgundy and Ube2N in green. Hydrogen-bonding interactions are given in black and hydrophobic interactions in red. d Comparison of the ubiquitinating activity of wild-type MavC versus mutant proteins using Ube2N-SS-Ub as the substrate. Reactions were subjected to SDS-PAGE and visualized with Coomassie Blue. A control reaction without MavC is included. e MavC-mediated ubiquitination of Ube2N during L.p infection. Cells infected with the indicated L. pneumophila strains were lysed with 0.2% saponin and lysate separated by SDS-PAGE, probed by immunoblotting with antibodies specific for Ube2N (upper panel) and MavC (lower panel). respectively. f The effects of MavC and its mutants on NFkappaB activation. HEK293T cells were transfected with plasmids expressing a luciferase reporter responsive to NF-kappaB and Flag-MavC or its mutant. At the same time, a plasmid expressing Renilla luciferase used as an internal control and stimulator TRAF6 were co-transfected. NF-kappaB activity was determined by dual luciferase assay. The expression of MavC and its mutants was probed in lysates of transfected cell while tubulin was detected as a loading control. Three independent experiments were done with similar results. Error bars indicate standard error of the mean (SEM).

Supportive validation

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Experimental details: 5 Fig. UCA1 inhibition releases miR-27a-5p which downregulates UBE2N and induces BIM expression. (A) Relative transcript distribution of UCA1 short and long isoforms (RT-qPCR) in nuclear and cytoplasmic compartments in OAW42-R cells normalized to total cellular transcript levels. NEAT1 was used as a control for nuclear transcript. (B) Venn diagram of overlapping miRNAs between miRNAs reported/predicted to bind UCA1 (left) and miRNAs whose seed is enriched in DEGs (right) after UCA1 downregulation in OAW42-R cells. (C) Schematic representation of short isoform of UCA1 and miR-27a-5p sequence complementarity as predicted by RNA22 v2 software. (D) Fold enrichment of UCA1 after Biotin-miR-27a-5p pull-down relative to Biotin-Cel-miR-67 pull-down. Data are expressed as mean +- SEM from 3 experiments. (E, G and H) BIM, PUMA, or UBE2N (unmodified and ubiquitinated) protein levels (western blot), quantified relative to actin loading control, representative or mean +- SEM of 3 experiments, after indicated siRNAs transfection in OAW42-R cells. (F) Pictures from cell layer. Data are representative of 3 experiments

Supportive validation

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Experimental details: Figure 1 E2 enzymes stimulate OTUB1 DUB activity ( a ) FRET-based assay for cleavage of internally quenched fluorescent K48 Ub 2 (400 nM) by OTUB1 (30 nM) in the absence and presence of 11 different E2 enzymes (5 uM). ( b ) The rates of K48 Ub 2 (400 nM) cleavage by OTUB1 (30 nM) are plotted as a function of the log of the concentration of UBCH5B. The data were analyzed and fit using a ""log(agonist) vs. response"" model to determine EC 50 . Each rate was measured in triplicate, and error bars represent the SEM for each measurement. ( c ) Coomassie stained gel showing K48 Ub 2 (15 uM) cleavage by OTUB1 (0.5 uM) in the presence and absence of UBCH5B (25 uM). ( d ) Steady-state kinetic saturation curve for cleavage of K48 Ub 2 by OTUB1 (30 nM) in the presence (red) and absence (blue) of UBCH5B (10 uM). Each rate was measured in triplicate, and error bars represent the SEM for each measurement.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 E2 enzymes exist in both charged and uncharged states in vivo ( a ) Western blot of cell lysates from HeLa and U2OS cells prepared at acidic pH (+- beta-ME) and probed with anti-UBC13 and anti-UBCH5 antibodies exhibit both charged and uncharged forms of the E2 enzymes. ( b ) Depiction of three possible states of OTUB1-E2 complexes. Top: OTUB1 (green) bound to K48 Ub 2 (orange) and E2 (blue) or E2 (blue)~Ub (purple) thioester. Bottom: OTUB1 bound to free Ub (yellow) and E2~Ub thioester. The free ubiquitin (yellow) and the ubiquitin (purple) conjugated to the E2 bind in place of K48 Ub 2 .