Anti-LATS1/WARTS antibody

• IP

Unknown

Immunoprecipitation - Anti-LATS1/WARTS antibody (AB70561)

Detection of human LATS1/WARTS by Immunoprecipitation in whole cell lysate from HeLa cells (1 mg, 1/4 of immunoprecipitate loaded/lane), using ab70561 at 3 μg/mg of lysate (lane 1).
LATS1/WARTS was also immunoprecipitated with an other LATS1/WARTS antibody (lanes 2) and with ab70562 (lane 3) using 3 μg/mg lysate.
Lane 4 : Control IgG immunoprecipitate.
Subsequent Western blot detection of LATS1/WARTS was performed using ab70561 at 0.1 μg/ml.

All lanes:

Immunoprecipitation - Anti-LATS1/WARTS antibody (ab70561)

Predicted band size: 126 kDa

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• WB

Unknown

Western blot - Anti-LATS1/WARTS antibody (AB70561)

All lanes:

Western blot - Anti-LATS1/WARTS antibody (ab70561) at 0.02 µg/mL

Lane 1:

Whole cell lysate from Hela cells at 50 µg

Lane 2:

Whole cell lysate from Hela cells at 15 µg

Predicted band size: 126 kDa

Observed band size: 117 kDa,160 kDa,200 kDa,268 kDa,71 kDa

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• WB

Supplier Data

Western blot - Anti-LATS1/WARTS antibody (AB70561)

All lanes:

Western blot - Anti-LATS1/WARTS antibody (ab70561) at 0.2 µg/mL

Lane 1:

CT26.WT whole cell lysate at 50 µg

Lane 2:

TCMK-1 whole cell lysate at 50 µg

Predicted band size: 126 kDa

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Exposure time: 3min

• WB

CiteAb

Western blot - Anti-LATS1/WARTS antibody (AB70561)

Western Blotting using Anti-LATS1/WARTS antibody, ab70561. 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.

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• WB

CiteAb

Western blot - Anti-LATS1/WARTS antibody (AB70561)

Western Blotting using Anti-LATS1/WARTS antibody, ab70561. Publication image from Chen, Y. et al., 2017, Nat Commun, 28474680. Legend direct from paper.

YAP plays a critical role in high-glucose-stimulated liver tumorigenesis.(a) Glucose inhibited YAP phosphorylation while stimulating YAP expression. Bel-7402 and SMMC-7721 cells were cultured in media containing the indicated concentrations of glucose for 24 h. YAP expression levels and phosphorylation were then measured by western blot. The levels of p-YAP were normalized to those of YAP, and the data from cells cultured in 5.5 mM glucose were arbitrarily set to 100%. (b,c) Glucose stimulated O-GlcNAcylation of YAP at Thr241. Bel-7402 and SMMC-7721 cells were cultured in media containing the indicated concentrations of glucose (b). Bel-7402 cells were transfected with WT and T241A YAP-FLAG, and cells were treated with glucose at the indicated final concentration for 24 h (c). Endogenous YAP or exogenous WT or T241A YAP-FLAG was immunoprecipitated with anti-YAP or anti-FLAG antibodies, and O-GlcNAcylation was measured by western blot using an anti-O-GlcNAc antibody. The levels of O-GlcNAcylation of endogenous YAP or exogenous YAP-FLAG were normalized to those of total YAP or YAP-FLAG, and data from cells cultured in 5.5 mM glucose were arbitrarily set to 100%. (d) Mutation of Thr241 induced LATS1-YAP binding under glucose stimulation. Expression plasmids of YAP-FLAG, WT or T241A were transfected into Bel-7402 and SMMC-7721 cells, and the cells were treated with the indicated final concentration of glucose for 24 h before analysis. YAP-FLAG was immunoprecipitated with anti-FLAG antibodies, and the association with LATS1 was measured by anti-LATS1 antibodies. (e–g) Knockdown of YAP abolished high-glucose-induced tumorigenesis. Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured by MTT-based assays (e), soft agar colony formation assays (f) and Caspase 3/7 Glo luciferase assays (g) in Bel-7402 and SMMC-7721 cells under the indicated treatments. Two shRNA sequences targeting YAP, sh1 and sh2, were used as indicated. Scale bar, 500 µm. (h) YAP and p-YAP were detected in the liver and colon of saline- and STZ-treated mice by western blot (left). O-GlcNAcylation of YAP in the liver from saline- and STZ-treated mice. YAP was immunoprecipitated with an anti-YAP antibody, and O-GlcNAcylation was detected with anti-O-GlcNAc antibodies using western blotting (middle). Silencing of YAP inhibited STZ-induced xenograft growth. Tumor volumes were monitored for 36 days after subcutaneous injection of different Bel-7402 cells as indicated; n=5 per group (right). Representative images are shown, and the data are expressed as the means+s.d. from three independent experiments (a–g). *P<0.05 and **P<0.01 indicate statistical significance. The data from a–c were analysed by one-way ANOVA, and the data from d–h were analysed by Student's t-test.

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