重组Anti-OGT / O-Linked N-Acetylglucosamine Transferase抗体[EPR12713]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Immunocytochemistry/ Immunofluorescence analysis of A549 (Human lung carcinoma epithelial cell) cells labeling OGT / O-Linked N-Acetylglucosamine Transferase using ab177941. The cells were fixed with 4% paraformaldehyde then permeabilized with 0.1% Triton X-100. The cells were then incubated with ab177941 at 1 : 100 dilution followed by a further incubation with a Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI. Cells were counterstained using ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1 : 200 dilution (shown in red). Secondary antibody only control : PBS instead of the primary antibody.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Immunohistochemical analysis of Paraffin-embedded sections human breast carcinoma tissue labelling OGT / O-Linked N-Acetylglucosamine Transferase with ab177941 at 1 : 2000 dilution (0.2 μg/ml), followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on human breast carcinoma tissue is observed. Counter stained with Haematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Immunohistochemical analysis of Paraffin-embedded sections human lung carcinoma tissue labelling OGT / O-Linked N-Acetylglucosamine Transferase with ab177941 at 1 : 2000 dilution (0.2 μg/ml), followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on human lung carcinoma tissue is observed. Counter stained with Haematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling OGT / O-Linked N-Acetylglucosamine Transferase with purified ab177941 at 1 : 40 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as a isotype control. Cell without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• WB

Lab

Western blot - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control.

All lanes:

Western blot - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (ab177941) at 1/10000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

Mouse heart at 20 µg

Lane 5:

Mouse pancreas at 20 µg

Lane 6:

Rat heart at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 116 kDa

Observed band size: 110 kDa

false

• WB

CiteAb

Western blot - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Western Blotting using Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713], ab177941. Publication image from Chen, Y. et al., 2017, Nat Commun, 28474680. Legend direct from paper.

YAP is O-GlcNAcylated by OGT at Thr241.(a) YAP O-GlcNAcylation was measured via HRP-labelled Streptavidin (left) and anti-TAMRA antibodies (middle) in Bel-7402 and SMMC-7721 cells. In vitro O-GlcNAcylation of YAP was measured by incubating purified YAP and OGT proteins with UDP-GlcNAc at 37 °C for 90 min before immunoprecipitation with anti-YAP antibodies, followed by western blot using an anti-O-GlcNAc antibody (right). (b) Enzymatic labelling of O-GlcNAc in WT and T241A YAP-FLAG proteins as analysed by anti-TAMRA antibodies and HRP-labelled Streptavidin in Bel-7402 and SMMC-7721 cells. (c) YAP-FLAG was immunoprecipitated by an anti-FLAG antibody in Bel-7402 and SMMC-7721 cells transfected with WT or T241A YAP-FLAG-expressing plasmids. Phosphorylation of YAP was detected using an anti-p-YAP antibody. (d) Mutation of Thr241 induced LATS1-YAP binding under stimulation of O-GlcNAcylation by combined treatment of GlcNAc (4 mM) and PuGNAc (25 µM). Expression plasmids for YAP-FLAG, WT or T241A were transfected into Bel-7402 and SMMC-7721 cells, and cells were treated as indicated for 24 h before analysis. YAP-FLAG was immunoprecipitated with anti-FLAG antibodies, and the association with LATS1 was measured using anti-LATS1 antibodies. (e) Mutation of Thr241 reduced YAP-promoted transformative phenotypes in vitro. Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured with MTT-based assays, soft agar colony formation assays and Caspase 3/7 Glo luciferase assays, respectively, in Bel-7402 and SMMC-7721 cells expressing WT or T241A YAP-FLAG. Scale bar, 500 µm. (f) Mutation of Thr241 diminished YAP-promoted xenograft tumour formation in vivo. The tumour size was measured 72 days after injection of Bel-7402 or SMMC-7721 cells into nude mice. (g) Mutation of Thr241 reduced the half-life of YAP. YAP-FLAG (as indicated) was transfected into Bel-7402 and SMMC-7721 cells, and cells were harvested at the indicated time points after addition of CHX (50 µg ml−1). The expression of YAP-FLAG was normalized to that of GAPDH, and the ‘0 h' point was arbitrarily set to 100%. Representative images are shown, and the data are expressed as the means+s.d. from three independent experiments. **P<0.01 indicates statistical significance. The data from d–f were analysed by Student's t-test.

false

• WB

CiteAb

Western blot - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Western Blotting using Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713], ab177941. Publication image from Chen, Y. et al., 2017, Nat Commun, 28474680. Legend direct from paper.

O-GlcNAcylation of YAP stimulates tumorigenesis.(a–d) Ectopic expression of YAP rescued the diminished transformative phenotypes caused by OGT knockdown in vitro. The protein expression of OGT, O-GlcNAc, p-YAP and YAP in Bel-7402 and SMMC-7721 cells was measured by WB (a). Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured with an MTT-based assay (b), soft agar colony formation assay (c) and Caspase 3/7 Glo luciferase assay (d), respectively, in Bel-7402 and SMMC-7721 cells under different treatments as indicated. Scale bar, 500 µm. (e,f) Ectopic expression of YAP rescued the xenograft tumour growth inhibited by OGT silencing in vivo. The protein expression of OGT, O-GlcNAc and YAP in xenografts was measured by WB (e). Tumour volumes were monitored for 36 days after subcutaneous injection of Bel-7402 cells into nude mice; n=5 per group (f). Representative IHC images of cleaved Caspase 3 and Ki67 staining in the xenografts formed in nude mice. Scale bar, 500 µm. Representative images from three independent experiments are shown (a–e). **P<0.01 indicates statistical significance. The data from b–d,f were analysed by one-way and two-way ANOVA, respectively.

false

• WB

CiteAb

Western blot - Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713] (AB177941)

Western Blotting using Anti-OGT / O-Linked N-Acetylglucosamine Transferase antibody [EPR12713], ab177941. Publication image from Chen, Y. et al., 2017, Nat Commun, 28474680. Legend direct from paper.

O-GlcNAcylation of YAP stimulates tumorigenesis.(a–d) Ectopic expression of YAP rescued the diminished transformative phenotypes caused by OGT knockdown in vitro. The protein expression of OGT, O-GlcNAc, p-YAP and YAP in Bel-7402 and SMMC-7721 cells was measured by WB (a). Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured with an MTT-based assay (b), soft agar colony formation assay (c) and Caspase 3/7 Glo luciferase assay (d), respectively, in Bel-7402 and SMMC-7721 cells under different treatments as indicated. Scale bar, 500 µm. (e,f) Ectopic expression of YAP rescued the xenograft tumour growth inhibited by OGT silencing in vivo. The protein expression of OGT, O-GlcNAc and YAP in xenografts was measured by WB (e). Tumour volumes were monitored for 36 days after subcutaneous injection of Bel-7402 cells into nude mice; n=5 per group (f). Representative IHC images of cleaved Caspase 3 and Ki67 staining in the xenografts formed in nude mice. Scale bar, 500 µm. Representative images from three independent experiments are shown (a–e). **P<0.01 indicates statistical significance. The data from b–d,f were analysed by one-way and two-way ANOVA, respectively.

false