Anti-MYL12A (phospho S19) 抗体

• WB

Supplier Data

Western blot - Anti-MYL12A (phospho S19) antibody (AB2480)

All lanes:

Western blot - Anti-MYL12A (phospho S19) antibody (ab2480) at 1/1000 dilution

Lane 1:

Regulatory Light Chain Non-Phospho recombinant protein, 50 ng

Lane 2:

Regulatory Light Chain Phospho recombinant protein, 50 ng

Lane 3:

Smooth Muscle Non-Phospho recombinant protein, 50 ng

Lane 4:

Smooth Muscle Phospho recombinant protein, 50 ng

Secondary

All lanes:

Peroxidase rabbit secondary antibody at 1/40000 dilution

Predicted band size: 20 kDa

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• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MYL12A (phospho S19) antibody (AB2480)

IHC-P of ab2480 at 2.5 μg/ml staining both vascular and myometrial smooth muscle cells of the human uterus. The image shows localization of the antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain.

• WB

Unknown

Western blot - Anti-MYL12A (phospho S19) antibody (AB2480)

Rabbit polyclonal to phospho Myosin Light Chain phospho Myosin Light Chain (Ser 20) (ab2480) used at a 1/5000 dilution to detect myosin light chain by Western blot. Either 13 ug (lanes 1-3) or 20 ug (lanes 4-7) of a mouse cardiac myocyte lysate was loaded on a 4-20% Criterion gel for SDS-PAGE. Samples were either mock-treated or CLA treated :
Lane 1 : untreated 45 min
Lane 2 : CLA 50 nm 45 min
Lane 3 : CLA 100 nm 45 min
Lane 4 : A21 untreated 45 min
Lane 5 : A22 A23187 5 min
Lane 6 : A23 A23187 15 min
Lane 7 : A24 A23187 60 min
After washing, a 1/5,000 dilution of anti-rabbit HRP (ab7090) was used as secondary.

Rabbit polyclonal to phospho Myosin Light Chain phospho Myosin Light Chain (Ser 20) (ab2480) used at a 1/5000 dilution to detect myosin light chain by Western blot. Either 13 ug (lanes 1-3) or 20 ug (lanes 4-7) of a mouse cardiac myocyte lysate was loaded on a 4-20

All lanes:

Western blot - Anti-MYL12A (phospho S19) antibody (ab2480)

Predicted band size: 20 kDa

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• WB

CiteAb

Western blot - Anti-MYL12A (phospho S19) antibody (AB2480)

Western Blotting using Anti-MYL12A (phospho S19) antibody, ab2480. Publication image from Cervero, P. et al., 2018, Nat Commun, 29410425. Legend direct from paper.

LSP1 and supervillin interact with a similar subset of myosin IIA regulators. a Western blots of macrophage lysates progressively extracted by increased concentration of TritonX-100 (1–5%), or in RIPA buffer. Note that LSP1, and also myosin IIA, are mostly extracted in the 2% and RIPA fractions. b Western blot of immunoprecipitation of endogenous LSP1 or myosin IIA from macrophage lysates, with control IgG and input. Blots were probed with indicated antibodies. Note cross-coprecipitation of LSP1 and myosin IIA, accompanied by coprecipitation of myosin regulators MLCK and calmodulin. Molecular weight in kDa is indicated. c Western blots of anti-GFP immunoprecipitation of lysates from macrophages expressing LSP1 full length or LSP1 domains fused to GFP (For inputs, see Supplementary Fig. 7A). Blots were probed with indicated antibodies. Note coprecipitation of myosin IIA with full length LSP1, and also with the C-terminal and the villin headpiece-like domains (V1V2) of LSP1. Interestingly only the full length and the C-terminal constructs, but not V1V2, are also able to bind MLCK, pMLC and calmodulin. An LSP1-positive band in the lane of the C-terminal construct probably reflects the fact that the anti-LSP1 antibody recognizes an epitope in the C-terminal half of LSP1. d Immunoprecipitation of myosin IIA from macrophage lysates, with addition of Mg2+/ATP and/or latrunculin A (10 µM), as indicated. Upper panel : colloidal Coomassie stained SDS PAGE gel, lower panels : corresponding western blots developed with indicated antibodies. Molecular weight is indicated in kDa. e Quantification of coprecipitated amounts of actin and LSP1, normalized to precipitations performed without addition of Mg2+/ATP and/or latrunculin A. f Western blots of anti-GFP immunoprecipitations from lysates of macrophages expressing LSP1-GFP or supervillin construct SV1-830-GFP developed with indicated primary antibodies. Molecular weight in kDa is indicated. Note that LSP1 and SV1-830 coprecipitate comparable amounts of myosin IIA, MLCK and calmodulin, but that supervillin-coprecipitated myosin is more activated, as indicated by pMLC signal

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• WB

CiteAb

Western blot - Anti-MYL12A (phospho S19) antibody (AB2480)

Western Blotting using Anti-MYL12A (phospho S19) antibody, ab2480. Publication image from Cervero, P. et al., 2018, Nat Commun, 29410425. Legend direct from paper.

LSP1 and supervillin interact with a similar subset of myosin IIA regulators. a Western blots of macrophage lysates progressively extracted by increased concentration of TritonX-100 (1–5%), or in RIPA buffer. Note that LSP1, and also myosin IIA, are mostly extracted in the 2% and RIPA fractions. b Western blot of immunoprecipitation of endogenous LSP1 or myosin IIA from macrophage lysates, with control IgG and input. Blots were probed with indicated antibodies. Note cross-coprecipitation of LSP1 and myosin IIA, accompanied by coprecipitation of myosin regulators MLCK and calmodulin. Molecular weight in kDa is indicated. c Western blots of anti-GFP immunoprecipitation of lysates from macrophages expressing LSP1 full length or LSP1 domains fused to GFP (For inputs, see Supplementary Fig. 7A). Blots were probed with indicated antibodies. Note coprecipitation of myosin IIA with full length LSP1, and also with the C-terminal and the villin headpiece-like domains (V1V2) of LSP1. Interestingly only the full length and the C-terminal constructs, but not V1V2, are also able to bind MLCK, pMLC and calmodulin. An LSP1-positive band in the lane of the C-terminal construct probably reflects the fact that the anti-LSP1 antibody recognizes an epitope in the C-terminal half of LSP1. d Immunoprecipitation of myosin IIA from macrophage lysates, with addition of Mg2+/ATP and/or latrunculin A (10 µM), as indicated. Upper panel : colloidal Coomassie stained SDS PAGE gel, lower panels : corresponding western blots developed with indicated antibodies. Molecular weight is indicated in kDa. e Quantification of coprecipitated amounts of actin and LSP1, normalized to precipitations performed without addition of Mg2+/ATP and/or latrunculin A. f Western blots of anti-GFP immunoprecipitations from lysates of macrophages expressing LSP1-GFP or supervillin construct SV1-830-GFP developed with indicated primary antibodies. Molecular weight in kDa is indicated. Note that LSP1 and SV1-830 coprecipitate comparable amounts of myosin IIA, MLCK and calmodulin, but that supervillin-coprecipitated myosin is more activated, as indicated by pMLC signal

false

• WB

CiteAb

Western blot - Anti-MYL12A (phospho S19) antibody (AB2480)

Western Blotting using Anti-MYL12A (phospho S19) antibody, ab2480. Publication image from Schipper, K. et al., 2019, Nat Commun, 31444332. Legend direct from paper.

Actomyosin relaxation enables survival upon E-cadherin loss. a Merged brightfield and GFP images of MMECs isolated from Wcre;mTmG and Wcre;Cdh1F/F;mTmG female mice in the absence or presence of 10 µM Y-27632. Asterisks indicate areas of zoom, red dotted lines display outgrowth of E-cadherin-deficient MMECs. Zooms display altered cell adhesion in the absence or presence of Y-27632 of GFP-positive E-cadherin-deficient cells derived from Wcre;Cdh1F/F;mTmG female mice. Scale bar, 50 µm. b Western blot analysis of Wcre;mTmG and Wcre;Cdh1F/F;mTmG MMECs stained for E-cadherin and tubulin (loading control). c Western blot analysis of subclones derived from the Wcre;mTmG and Wcre;Cdh1F/F;mTmG MMECs stained for E-cadherin and tubulin (loading control). d Brightfield images of MMECs 3 h post seeding in the absence or presence of 10 µM Y-27632 or 10 µM blebbistatin. Scale bar, 20 µm. e Western blot analysis of pSer473 Akt, Akt, cleaved caspase-3, and actin (loading control) in Wcre;Cdh1F/F;mTmG subclones grown in the absence or presence of 10 µM Y-27632. f, g Representative images (f) and quantification (g) of clonogenic assays with Wcre;mTmG (circles) and Wcre;Cdh1F/F;mTmG (squares) subclones grown in the presence or absence of 10 µM Y-27632 or 10 µM blebbistatin (7 days after seeding the cells). Data are of three independent experiments with three clones per experiment. h Western blot analysis of WT FVB and Cdh1δ/δ MMECs stained for E-cadherin and actin (loading control). i, j Representative images (i) and quantification (j) of clonogenic assays with Cdh1δ/δ and WT control MMEC lines with or without 10 µM Y-27632. Data are of three independent experiments. k Representative images of immunoblots of active RhoA pull-down assays and ser19 myosin light chain phosphorylation (pMLC) from Cdh1δ/δ and WT control MMECs harvested 3 h post seeding. l Quantification of active RhoA pull-down assays by densitometry normalized to the actin loading control. Data are of three independent experiments. All data are depicted as mean ± standard deviation. All p values were calculated using an unpaired two tailed t test. Source data are provided as a Source Data file

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