Antibody staining was performed as previously described (Lepage et al., 2014 (link)). Dilutions were as follows: rhodamine-phalloidin (1:200), anti-E-cadherin (Abcam, 1:1000), anti-RAB11B (Abcam 1:200), anti-ZO-1 (ThermoFisher Scientific, 1:500), anti-phospho-myosin-light chain 2 Ser 19 (Cell Signaling, 1:100). Embryos were mounted in either 80% glycerol or 0.05% low-melt agarose. Secondary antibodies used were goat-anti-mouse Alexa 488 (Invitrogen A11001, 1:500), goat anti-rabbit Alexa 488 (Invitrogen A11008, 1:500), goat anti-rabbit-Cy3 (Jackson ImmunoResearch AB-2338006,1:500). Sytox green (Invitrogen) was diluted to 0.5 mM in fixative. For embryos co-labelled with phalloidin and antibody, phalloidin was incubated at 1:200 dilution with primary antibody overnight at 4oC in block solution.
Anti phospho myosin light chain 2 ser19
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
The Anti-phospho Myosin Light Chain 2 (Ser19) is a specific antibody that recognizes the phosphorylated form of myosin light chain 2 at serine 19. This antibody is designed for the detection and analysis of phosphorylated myosin light chain 2 in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Imagexpress micro, by Molecular Devices (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rhodamine phalloidin, by Abcam (1 mentions)
Anti rab11b, by Abcam (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti pfak y397, by Cell Signaling Technology (1 mentions)
Anti fak, by BD (1 mentions)
Anti actin ab 5, by BD (1 mentions)
Anti ttl, by Proteintech (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Biotium (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Leica dmi8 epifluorescence microscope, by Leica Microsystems (1 mentions)
Clara cooled ccd camera, by Oxford Instruments (1 mentions)
Anti β catenin, by BD (1 mentions)
11 protocols using anti phospho myosin light chain 2 ser19
1
Comprehensive Antibody Staining Protocol
2
Immunofluorescence Staining of Glu-Tubulin
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Antibodies used were the following: anti-Glu-tubulin (Abcam, cat# ab48389, 1/250 dilution for IF, and 1/1000 for WB); anti-α-tubulin (Sigma-Aldrich, cat# T6199, 1/1000 dilution); anti-actin (Ab-5, BD Biosciences, cat# 612656, 1/4000 dilution); anti-FAK (BD Biosciences, cat# 610088, 1/250 dilution); anti-pFAK(Y397) (Cell Signaling Technology, cat# 3283 S, 1/250 dilution); anti-phospho-myosin light chain 2 (Ser 19) (Cell Signaling Technology, cat# 3671, 1/200 dilution); anti-myosin (clone MY-21, Sigma cat# M4401, 1/250 dilution); anti-TTL (Proteintech cat# 13618-AP, 1/500 dilution). Images of uncropped WBs are shown in Supplementary Fig. 10 .
For IF, cells were plated onto gels and were fixed with 4%PFA in PBS (phosphate-buffered saline) for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked with 3% BSA for 1 h, and incubated with primary antibodies overnight at 4 °C. Secondary antibodies were conjugated with Alexa 488, 546, or 647 (Alexa Fluora series from Thermo Fisher Scientific). Nuclei or whole-cell area was stained with DAPI or cell mask blue stain, respectively. For determination of percentage of cells with a Glu-MT network, positive cells were defined as those having > 10–15 distinctly stained Glu-MT fibers.
For IF, cells were plated onto gels and were fixed with 4%PFA in PBS (phosphate-buffered saline) for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked with 3% BSA for 1 h, and incubated with primary antibodies overnight at 4 °C. Secondary antibodies were conjugated with Alexa 488, 546, or 647 (Alexa Fluora series from Thermo Fisher Scientific). Nuclei or whole-cell area was stained with DAPI or cell mask blue stain, respectively. For determination of percentage of cells with a Glu-MT network, positive cells were defined as those having > 10–15 distinctly stained Glu-MT fibers.
3
Immunofluorescence Imaging of Cytoskeleton Proteins
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Cells
were permeabilized and fixed in buffer C (10 mM MES (2-morpholinoethanesulfonic
acid), 3 mM MgCl2, and 138 mM KCl (pH 6.8) with 4% paraformaldehyde,
1.5% (w/v) bovine serum albumin, and 0.5% (v/v) Triton-X for 15 min.
The cells were incubated overnight at 4 °C with primary antibodies
and for 1 h at room temperature with secondary antibodies. Primary
antibodies used were anti-β-catenin (Clone 14, 610,154, BD Biosciences,
San Jose, CA), anti-paxillin (Y113, ab32084, Abcam, Cambridge, UK),
anti-phosphomyosin Light Chain 2 (Ser 19) (3671S, Cell Signaling Technology,
Danvers, MA), and anti-E-cadherin (DECMA-1, sc-59778, Santa Cruz Biotechnology,
Dallas, TX and CM1681, ECM Biosciences, Versailles, KY). Alexa Fluor
488-conjugated phalloidin was from Thermo Fisher Scientific, Waltham,
MA, and DAPI was from Biotium, Hayward, CA. Secondary antibodies were
from Jackson ImmunoResearch, West Grove, PA. Jasplakinolide (used
at 1 nM for 1 h) and SMIFH2 (used at 20 μM for 2 h) were from
MilliporeSigma, Burlington, MA. All images were taken using a Leica
DMi8 epifluorescence microscope (Leica Microsystems, Buffalo Grove,
IL) with 10×, 20×, or 40× objectives and a Clara cooled
CCD camera (Andor Technology, Belfast, Northern Ireland).
were permeabilized and fixed in buffer C (10 mM MES (2-morpholinoethanesulfonic
acid), 3 mM MgCl2, and 138 mM KCl (pH 6.8) with 4% paraformaldehyde,
1.5% (w/v) bovine serum albumin, and 0.5% (v/v) Triton-X for 15 min.
The cells were incubated overnight at 4 °C with primary antibodies
and for 1 h at room temperature with secondary antibodies. Primary
antibodies used were anti-β-catenin (Clone 14, 610,154, BD Biosciences,
San Jose, CA), anti-paxillin (Y113, ab32084, Abcam, Cambridge, UK),
anti-phosphomyosin Light Chain 2 (Ser 19) (3671S, Cell Signaling Technology,
Danvers, MA), and anti-E-cadherin (DECMA-1, sc-59778, Santa Cruz Biotechnology,
Dallas, TX and CM1681, ECM Biosciences, Versailles, KY). Alexa Fluor
488-conjugated phalloidin was from Thermo Fisher Scientific, Waltham,
MA, and DAPI was from Biotium, Hayward, CA. Secondary antibodies were
from Jackson ImmunoResearch, West Grove, PA. Jasplakinolide (used
at 1 nM for 1 h) and SMIFH2 (used at 20 μM for 2 h) were from
MilliporeSigma, Burlington, MA. All images were taken using a Leica
DMi8 epifluorescence microscope (Leica Microsystems, Buffalo Grove,
IL) with 10×, 20×, or 40× objectives and a Clara cooled
CCD camera (Andor Technology, Belfast, Northern Ireland).
4
Immunofluorescence Analysis of Drosophila Genital Disc
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Pupae selection, genital disc dissection, and immunofluorescence were carried out as described elsewhere (Kuckwa et al., 2016 (link)) and were repeated at least three times. The following antibodies were used: anti Duf/Kirre (1:500, Kreisköther et al., 2006 (link)), anti-Cadherin-N (1:100, DSHB DN-Ex #8), anti-Shotgun (1:100, DSHB DCAD2), anti-Stumps (Dof, 1:1000; gift from Maria Leptin, University of Cologne, Germany; Vincent et al., 1998 (link)), antiSqh [1:10, 64 h incubation time; anti Phospho-Myosin Light Chain 2 (ser19), #3671 Cell Signaling; dilution according to Saxena et al., 2014 (link); Nie et al., 2014 (link)] and anti-Trol (Perlecan domain V, 1:2000; gift from Stefan Baumgartner, Lund University, Sweden; Friedrich et al., 2000 (link)). The following secondary antibodies were used: anti-rat Cy3 (1:500; Jackson ImmunoResearch Laboratories), anti-rat Alexa Fluor® 488 (1:500; Jackson ImmunoResearch Laboratories), anti-rabbit DyLight 488 (1:500; Vector Laboratories), anti-rabbit DyLight 549 (1:500; Vector Laboratories). For visualization of F-actin, we used Phalloidin-Atto 565 (4 nmol l−1; 94072, Sigma-Aldrich, St. Louis, MO, USA); to visualize nuclei, Hoechst 33342 was used (3.2 µg ml−1; 62249, Thermo Fisher Scientific, Waltham, MA, USA).
5
Immunolabeling and Microscopy of Embryos
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Embryos were fixed in 2% paraformaldehyde in seawater for 1 hr then washed 4 times in PBST, 10 min each. Embryos were incubated in PBST + 5% goat serum for 1 hr at room temperature, or overnight at 4°C, with the primary antibody diluted 1:1000 (anti-GFP, anti-myc, anti-RFP; Invitrogen, Grand Island, NY), 1:650 (anti-HA; Millipore, Billerica, MA), 1:300 (anti-PKC ζ; Santa Cruz Biotechnology, Dallas, TX), or 1:250 (anti-phospho-Myosin Light Chain 2 [Ser19]; Cell Signaling Technology, Danvers, MA). Animals were washed 5× for 10 min in PBST and then placed in an appropriate secondary antibody, and incubated as described for the primary antibody. Secondary antibodies used were anti-mouse or anti-rabbit Alexa Fluor-labeled antibodies (Invitrogen) with a range of excitation/emission spectra, depending on the experiment. Samples were washed 4 to 10× in PBST following secondary incubation. For microscopy, labeled embryos were immobilized onto cover slips coated with 0.08% Poly-L-lysine. Fixed embryos were cleared in 80% glycerol or in an isopropyl alcohol series followed by 2:1 benzyl alcohol:benzoyl benzoate (BABB).
6
Quantifying phosphorylated Myosin Light Chain
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After fixation in PBS containing 4% formaldehyde, cells were washed three times in PBS, permeabilized and blocked with 0.1% Triton X-100, 1% BSA, 10% FBS, and 0.01% NaN3 for 1 hr at 25 °C, and stained overnight at 4 °C with anti-phospho Myosin Light Chain 2 (Ser19) (Cell Signaling Technology, 3671, 1:200). Primary antibody was visualized using Alexa Fluor 488 (Life Technology, A11034, 1:1,000). Images were taken on an ImageXpress Micro (Molecular Devices) with 20X (0.75 N.A.) objective.
7
Immunofluorescence Analysis of Endothelial Cells
8
Immunostaining Embryonic Tissue Samples
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Embryos were fixed in 4% paraformaldehyde with 1% DMSO in 0.1 M phosphate buffer, pH 7.4, then dehydrated in methanol and permeabilized for 30 min. in acetone at -20°C or 1mg/ml collagenase for 7 min (NCadh), and rehydrated with incubation buffer (0.2% BSA, 0.5% Triton X 100 in 0.1 M phosphate buffer, pH 7.4). The following primary antibodies were used: anti-SV2 antibody (1:50, Developmental Studies Hybridoma Bank [DSHB]), anti-phospho-myosin light chain 2 (Ser19, 1:20, Cell Signaling Technology), anti-c-myc clone 9E1 (1:600, Sigma) and anti-F59 (1:10, DSHB) [56 (link), 57 (link)], anti-En1 clone 4D9 (1:5, DSHB), anti-NCadh (1:100, Abcam ab12221), anti-znp1 (1:200) [58 (link)], and for labeling of AChR, we used Alexa 594-coupled α-bungarotoxin (10μg/ml, Molecular Probes). Embryos were washed at least three times in incubation buffer before adding secondary antibodies conjugated with Alexa Fluor 488, 568 or 594 (1:400, Life Technologies). Antibody incubations were performed for 4 h at room temperature or overnight at 4°C. Embryos were mounted in Vectashield mounting medium (Vector laboratories), head tissue was used for genotyping (see below) and samples were viewed and documented as described below.
9
Quantifying phosphorylated Myosin Light Chain
10
Quantitative Western Blot Analysis of Protein Expression
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To determine protein expression by western blot, HUVECs were washed, lysed in RIPA buffer containing phosphatase and protease inhibitors (200 μM pefablock 0.8 μM aprotinin, 11 μM leupeptin, 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma‐Aldrich]) for 45 min on ice, and centrifuged to remove cell debris. The protein concentration of the cell lysates was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol. 25 μg of protein of each sample was separated on 8% SDS–polyacrylamide gels and transferred on nitrocellulose membranes by semi‐dry blotting. The membranes were blocked in 3% BSA for 1 h at RT and incubated with VE‐cadherin (eBioscience), anti‐phospho‐myosin light chain2 (Ser19) (Cell Signaling Technology), anti‐α‐tubulin (Cell Signaling), or anti‐GAPDH (Sigma‐Aldrich) overnight at 4°C. Membranes were then incubated with anti‐rabbit IgG‐HRP (Cell Signaling) or anti‐mouse IgG‐HRP (Cell Signaling) for 1 h at RT. The Clarity western ECL Blotting Substrate (BIO RAD) was used according to the manufacturer's protocol for signal development and chemiluminescence was detected using the Fusion FX chemiluminescence imager (Vilber Lourmat, Marne‐la‐Vallée, France). Densitometric quantification of blots was performed using ImageJ and protein levels were normalized to α‐tubulin or GAPDH.
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