Pthr18 ser19 mlc2

Manufactured by Cell Signaling Technology

Sourced in United Kingdom

PThr18/Ser19-MLC2 is a phospho-specific antibody that recognizes myosin light chain 2 (MLC2) phosphorylated at Thr18 and Ser19 residues. This antibody is designed for use in Western blotting and immunohistochemical applications to detect the phosphorylation of MLC2 at these specific sites.

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Lab products found in correlation

Gapdh, by Merck Group (5 mentions) Lamin b1, by Proteintech (2 mentions) Irdye 680rd goat anti rabbit igg, by LI COR (2 mentions) Lamin b2, by Proteintech (2 mentions) Irdye 800rd goat anti mouse igg, by LI COR (2 mentions) Odyssey fc, by LI COR (2 mentions) Py705 stat3, by Cell Signaling Technology (2 mentions) Lamin a c, by Proteintech (1 mentions) Lds buffer 1x, by Thermo Fisher Scientific (1 mentions) Mcl 1, by Santa Cruz Biotechnology (1 mentions) Pd l1 clone e1l3n, by Cell Signaling Technology (1 mentions) Gapdh mab374, by Merck Group (1 mentions) P h2a x s139, by Merck Group (1 mentions) Ncl l cd4 368, by Leica (1 mentions) Pser19 mlc2, by Cell Signaling Technology (1 mentions) F4 80, by Abcam (1 mentions) Cd206, by Abcam (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Tritc conjugated phalloidin, by Thermo Fisher Scientific (1 mentions) Huvecs, by Lonza (1 mentions)

6 protocols using pthr18 ser19 mlc2

Western Blot Analysis of Melanoma Cells

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Melanoma cells were seeded at a density of 6 × 10⁵ cells ml⁻¹ in 12-well plates and lysed the next day with LDS buffer 1× (Life Technologies). Lysates were denatured at 95 °C for 5 min and sonicated. SDS–PAGE and western blotting were performed using standard procedures. Membranes were visualized and bands quantified using an Odyssey Fc (LI-COR). Loading controls were run on the same blot. Individual 680 and 800 channels were co-visualized in greyscale on the same image. Primary antibodies were: LAP1 (1:1,000; #21459-1-AP), Lamin A/C (1:1,000, #10298-1-AP), Lamin B1 (1:1,000, #12987-1-AP), Lamin B2 (1:1,000, #10895-1-AP) from Proteintech; pThr18/Ser19-MLC2 (1:750, #3674), MLC2 (1:750, #3672) from Cell Signaling Technology; GAPDH (1:10,000, #MAB374) from Merck. Secondary antibodies were: IRDye 680RD goat anti-rabbit IgG (1:10,000, #925-68071) and IRDye 800RD goat anti-mouse IgG (1:10,000, #925-32210) from LI-COR.

Jung-Garcia Y., Maiques O., Monger J., Rodriguez-Hernandez I., Fanshawe B., Domart M.C., Renshaw M.J., Marti R.M., Matias-Guiu X., Collinson L.M., Sanz-Moreno V, & Carlton J.G. (2023). LAP1 supports nuclear adaptability during constrained melanoma cell migration and invasion. Nature Cell Biology, 25(1), 108-119.

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Quantification of Melanoma Cell Proteins

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Melanoma cells were seeded at a density of 6x10⁵ cells/ml in 12-well plates and lysed the next day with LDS buffer 1X (Life Technologies). Lysates were denatured at 95°C for 5 minutes and sonicated. SDS-PAGE and western blotting was performed using standard procedures. Membranes were visualised and bands quantified using an Odyssey Fc (LI-COR). Loading controls were run on the same blot. Individual 680 and 800 channels were co-visualised in greyscale on the same image. Primary antibodies were: LAP1 (1:1000; #21459-1-AP), Lamin A/C (1:1000, #10298-1-AP), Lamin B1 (1:1000, #12987-1-AP), Lamin B2 (1:1000, #10895-1-AP) from Proteintech; pThr18/Ser19-MLC2 (1:750, #3674), MLC2 (1:750, #3672) from Cell Signalling Technology; GAPDH (1:10,000, #MAB374) from Merck. Secondary antibodies were: IRDye 680RD goat anti-rabbit IgG (1:10,000, #925-68071) and IRDye 800RD goat anti-mouse IgG (1:10,000, #925-32210) from LI-COR.

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Immunoblot and Immunohistochemistry Analyses

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Antibodies and concentrations used: pThr18/Ser19-MLC2 (#3674; 1:750, immunoblot), pSer19-MLC2 (#3671; 1:50, immunohistochemistry; 1:200, immunofluorescence), MLC2 (#3672; 1:750), pT202/Y204-p44/42 (ERK1/2) (#4370; 1:1,000), pY705-STAT3 (#9145; 1:750), PD-L1 (clone E1L3N, #13684, 1:200) from Cell Signaling Technology; STAT3 (sc-482; 1:500), ERK2 (sc-154; 1:1,000), MCL-1 (sc-819; 1:1,000), GFP (sc-8334; 1:1,000) from Santa Cruz Biotechnology; GAPDH (MAB374; 1:10,000) from Millipore; P-H2A.X (S139) (ab2893;1:1000), CD206 (ab64693; 1:1,000), CD3 (anti-mouse, ab134096; 1:500), CD4 (anti-mouse, clone I3T4, ab183685; 1:300), FoxP3 (anti-human, clone 236A/E7, ab20034; 1:200) from Abcam; F4/80 (anti-mouse, clone BM8, MF48000, 1:1000), CD8a (anti-mouse, clone Ly2, 14-0808-82; 1:200), FoxP3 (anti-mouse, clone FJK-16s, 14-5773-82; 1:200) from Invitrogen; CD4 (anti-human, clone 11E9, NCL-L-CD4-368; 1:300) from Novocastra.

Orgaz J.L., Crosas-Molist E., Sadok A., Perdrix-Rosell A., Maiques O., Rodriguez-Hernandez I., Monger J., Mele S., Georgouli M., Bridgeman V., Karagiannis P., Lee R., Pandya P., Boehme L., Wallberg F., Tape C., Karagiannis S.N., Malanchi I, & Sanz-Moreno V. (2020). Myosin II Reactivation and Cytoskeletal Remodeling as a Hallmark and a Vulnerability in Melanoma Therapy Resistance. Cancer Cell, 37(1), 85-103.e9.

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Immunoblotting Protocols for HUVECs and PC3 Cells

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Primary human umbilical vein endothelial cells (HUVECs) (Lonza) and PC3 cells were grown as previously described (Reymond et al., 2012, 2012).
For western blotting, primary antibodies were used at a dilution of 1:1000 and secondary HRP‐conjugated mouse or rabbit antibodies (Amersham) at 1:5000. The following antibodies were used: RhoC (C‐16, Santa Cruz Biotechnology or D40E4, Cell Signalling), ROCK1 and ROCK2 (mouse, BD Transduction Laboratories), VE‐cadherin (clone 75, BD Biosciences), PECAM‐1 (clone JC70A, Dako), PE‐PECAM‐1 (clone 390, BioLegend), pThr18/Ser19‐MLC2 (#3674, Cell Signalling), MLC2 (#3672, Cell Signalling), and GAPDH (Millipore). HRP‐conjugated antibodies were detected with chemiluminescence reagent (Pierce). TRITC‐conjugated phalloidin (1:400; Invitrogen) were used to detect F‐actin. Where indicated, PC3 cells were labelled with 2 μM 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in RPMI containing 0.1% FCS.

Reymond N., Im J.H., Garg R., Cox S., Soyer M., Riou P., Colomba A., Muschel R.J, & Ridley A.J. (2015). RhoC and ROCKs regulate cancer cell interactions with endothelial cells. Molecular Oncology, 9(6), 1043-1055.

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Western Blot Analysis of Phospho-Proteins

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Cells were lysed in Laemmli sample buffer, boiled for 5 min, sonicated for 15 s and spun down. Lysates were fractionated using 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF filters (0.45 µm, Immobilon). ECL or Prime ECL detection Systems (GE Healthcare) with HRP-conjugated secondary antibodies (GE Healthcare) were used for detection. Bands were quantified using Image J. Levels of phospho-proteins were calculated after correction to total levels of the relevant protein. Antibodies: pThr18/Ser19-MLC2 (1:750, #3674), MLC2 (1:750, #3672) and RhoA (1:1000, #2117) from Cell Signalling Technology; GFP (1:10000, sc-8334) from Santa Cruz Biotechnology; GAPDH (1:10000, MAB374) from Millipore.

Rodriguez-Hernandez I., Maiques O., Kohlhammer L., Cantelli G., Perdrix-Rosell A., Monger J., Fanshawe B., Bridgeman V.L., Karagiannis S.N., Penin R.M., Marcolval J., Marti R.M., Matias-Guiu X., Fruhwirth G.O., Orgaz J.L., Malanchi I, & Sanz-Moreno V. (2020). WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion. Nature Communications, 11, 5315.

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Investigation of MMP-9 and MMP-13 Regulation

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Antibodies and dilutions used: MMP-9 (Clone 4H3, MAB911; 1:1,000), MMP-13 (MAB511; 1:500) from R&D Systems; pSer19-MLC2 (immunofluorescence; 1:200), pThr18/Ser19-MLC2 (immunoblotting, 1:750) and pY705-STAT3 (1:750) from Cell Signaling Technology (no. 3671, no. 3674 and no. 9145, respectively); STAT3 (sc-482; 1:500) and MLC2 (sc-15370; 1:200) from Santa Cruz Biotechnology; ROCK1 (611137; 1:1,000) from BD Transduction; GAPDH (MAB374; 1:10,000) from Millipore; CD44 (Clone IM7, ab119863; 1:2,000) from Abcam; MT1-MMP (SAB4501901; 1:1,000) from Sigma; affinity-purified antibody COL1-¾C (no. 0217-050, Immunoglobe; 1:50) directed against the C-terminal cleavage neo-epitope of collagen types I and II (refs 22 (link),31 (link)).
ProMMP-9 (2–4 μg ml ⁻¹, no. PF038), P6 (1–10 μM), H1152 (5 μM) and MMP-9 inhibitor I (0.05–1 μM) were from Calbiochem (Nottingham, UK); Y27632 (10 μM) from Tocris Bioscience (Bristol, UK).

Orgaz J.L., Pandya P., Dalmeida R., Karagiannis P., Sanchez-Laorden B., Viros A., Albrengues J., Nestle F.O., Ridley A.J., Gaggioli C., Marais R., Karagiannis S.N, & Sanz-Moreno V. (2014). Diverse matrix metalloproteinase functions regulate cancer amoeboid migration. Nature communications, 5, 4255.

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