Anti-Sumo 2 + Sumo 3 antibody [8A2]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Immunocytochemistry/immunofluorescence analysis of HeLa cells labelling Sumo 2 + Sumo 3 with ab81371 at 1/50 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Primary antibody, ab81371 at 1 : 50 was incubated overnight at 4° C, followed by AlexaFluor® 488-conjugated Goat anti- Mouse secondary antibody (ab150113) at 1/1000 dilution at RT for 45 min. ab179513 Anti-beta Tubulin, used as a counterstain at 1/200 dilution, was co-incubated with ab81371 overnight at 4° C, followed by Alexa Fluor® 594 Goat Anti-Rabbit. secondary (ab150080) at 1/1000 dilution at RT for 45 min. Nucleus were visualized using DAPI.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Immunocytochemistry/immunofluorescence analysis of SW480 cell line labelling Sumo 2 + Sumo 3 with ab81371 at 1/50 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Primary antibody, ab81371 at 1 : 50 was incubated overnight at 4° C, followed by AlexaFluor® 488-conjugated Goat anti- Mouse secondary antibody (ab150113) at 1/1000 dilution at RT for 45 min. ab179513 Anti-beta Tubulin, used as a counterstain at 1/200 dilution, was co-incubated with ab81371 overnight at 4° C, followed by Alexa Fluor® 594 Goat Anti-Rabbit. secondary (ab150080) at 1/1000 dilution at RT for 45 min. Nucleus were visualized using DAPI.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Sumo 2 + Sumo 3 with ab81371 at 1/1000 dilution followed by a ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879). The section was incubated with ab81371 for overnight at 4℃.
Secondary antibody only control : Ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879).
Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution1).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Immunocytochemistry/immunofluorescence analysis of NIH/3T3 cells labelling Sumo 2 + Sumo 3 with ab81371 at 1/50 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Primary antibody, ab81371 at 1 : 50 was incubated overnight at 4° C, followed by AlexaFluor® 488-conjugated Goat anti- Mouse secondary antibody (ab150113) at 1/1000 dilution at RT for 45 min. ab179513 Anti-beta Tubulin, used as a counterstain at 1/200 dilution, was co-incubated with ab81371 overnight at 4° C, followed by Alexa Fluor® 594 Goat Anti-Rabbit. secondary (ab150080) at 1/1000 dilution at RT for 45 min. Nucleus were visualized using DAPI.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Sumo 2 + Sumo 3 with ab81371 at 1/1000 dilution followed by a ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879). The section was incubated with ab81371 for overnight at 4℃.
Secondary antibody only control : Ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879).
Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution1).

• WB

Lab

Western blot - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Sumo 2 + Sumo 3 antibody [8A2] (ab81371) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate B276 at 10 µg

Lane 2:

SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate at 10 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 10 µg

Lane 4:

SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 10 µg

Lane 5:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 10 µg

Lane 6:

293T (human embryonic kidney epithelial cell), whole cell lysate at 10 µg

Lane 7:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg

Lane 8:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 12 kDa

Observed band size: 15 kDa

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• WB

Unknown

Western blot - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Dilution and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Sumo 2 + Sumo 3 antibody [8A2] (ab81371) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 10 µg

Lane 2:

Human kidney tissue lysate at 10 µg

Lane 3:

Human spleen tissue lysate at 10 µg

Lane 4:

Mouse brain tissue lysate at 10 µg

Lane 5:

Rat spleen tissue lysate at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 12 kDa

Observed band size: 15 kDa

false

• WB

CiteAb

Western blot - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Western Blotting using Anti-Sumo 2 + Sumo 3 antibody [8A2], ab81371. Publication image from Eifler, K. et al., 2018, Nat Commun, 29549242. Legend direct from paper.

Knockdown of the SUMO conjugation pathway delays transition through mitosis and induces the formation of chromosome bridges. a Knockdown of the SUMO-activating enzyme (SAE) in HeLa cells was achieved by stably expressing inducible shRNAs generated against both SAE subunits SAE1 (ishSAE1) and SAE2 (ishSAE2.1 and ishSAE2.2). Scrambled shRNA was used as a control (ishControl). These cells were analyzed by live cell microscopy 48 h after induction of the SAE knockdown. Pictures were acquired every 5 min and a selection of pictures is depicted here. Scale bars correspond to 10 µM. b The amount of time needed for cells to pass from nuclear envelope breakdown (NEB) to metaphase (dark grey) and from metaphase to anaphase (light grey) was quantified by live cell imaging. Standard deviations were calculated for >200 cells resulting from three independent experiments. A two-sided Student’s t-test was performed. *p-values < 0.05. c To confirm a reduction of SUMO conjugates and SAE2 expression, lysates of HeLa cells expressing inducible SAE knockdown constructs were analyzed by immunoblotting with anti-SUMO2/3 and anti-SAE2 antibody 48 h after induction and compared to the control. Equal loading of the lysates was verified by staining with Ponceau S. d HeLa cells expressing inducible SAE knockdown constructs were fixed 72 h after induction and stained with Hoechst to monitor the formation of chromosome bridges (white arrows). Scale bars correspond to 10 µM. e The percentage of cells with DNA bridges 72 h after induction of SAE knockdown was quantified using the microscopy approach described above, analyzing 100 cells per condition. The standard error of the mean was calculated from three independent experiments. A two-sided Student’s t-test was performed. *p-values < 0.005

false

• WB

CiteAb

Western blot - Anti-Sumo 2 + Sumo 3 antibody [8A2] (AB81371)

Western Blotting using Anti-Sumo 2 + Sumo 3 antibody [8A2], ab81371. Publication image from Eifler, K. et al., 2018, Nat Commun, 29549242. Legend direct from paper.

Posttranslational modification of the APC/C by SUMOylation. a Alignment of the Cullin domains present in the human Cullin proteins and the Cullin-like domain of APC2 : The NEDDylated lysine residue within the Cullin proteins is highlighted in red and is absent in APC2. b HeLa cells were treated with DMSO as a negative control (−) or 1 µM MLN4924 (+), an inhibitor of the NEDD8-activating enzyme, for 16 h. Lysates were analyzed by immunoblotting with anti-APC2 and anti-CUL4A antibodies. Ponceau S staining was performed to monitor equal loading. c HeLa cells and HeLa cells expressing His-tagged SUMO2 (His-SUMO2) were subjected to a His-pulldown (His-PD). Input and pulldown samples were analyzed by immunoblotting making use of anti-APC4 and anti-SUMO2/3 antibodies. Blots were stained with Ponceau S to monitor equal loading. At least three independent experiments were performed and representative results are shown

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