Phospho myosin light chain thr18 ser19

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-myosin light chain (Thr18/Ser19) is a lab equipment product that detects the phosphorylation of myosin light chain at threonine 18 and serine 19. This phosphorylation event is involved in the regulation of myosin activity and cellular processes such as muscle contraction and cell migration.

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Lab products found in correlation

Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Desmin, by Abcam (1 mentions) Phospho jnk, by Cell Signaling Technology (1 mentions) Gapdh, by Merck Group (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Catalase, by Abcam (1 mentions) Protease and phosphatase cocktail, by Thermo Fisher Scientific (1 mentions) Gradient gel, by Bio-Rad (1 mentions) Phospho and total erk1 2, by Cell Signaling Technology (1 mentions) Odyssey clx, by LI COR (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions)

3 protocols using phospho myosin light chain thr18 ser19

Measuring MLC20 Phosphorylation in Airway Smooth Muscle

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MLC₂₀ phosphorylation in BSM strips was measured as described
previously (28 (link)). Briefly, BSM strips
removed from silicone elastomer were suspended in organ baths containing Tyrode
buffer and stretched to generate a force of 1.5 g. Following equilibration, the
strips were stimulated with either high potassium (120 mM KCl) or carbachol (10
μM) and then frozen rapidly by clamp-freezing in liquid nitrogen,
followed by immersion in a dry ice/acetone slurry containing 10% trichloroacetic
acid (TCA) and 10 mM DTT. The strips were slowly thawed at room temperature,
washed in acetone, air dried, and then subjected to homogenization and
precipitated the protein in 10% TCA with 10 mM DTT. The samples were centrifuged
at 12,000 rpm for 10 min and the protein pellet was washed with diethylehter,
air-dried and resuspended in sample buffer. Equal amounts of proteins were
resolved on SDS-PAGE and transferred to polyvinylidene difluoride membranes
(Millipore, Bedford, MA). The proteins were subjected to immunoblot analysis
using phospho-myosin light chain (Thr18/Ser19) (Cell Signaling Technology,
Danvers, MA, USA) and myosin light chain antibodies (Abcam, Cambridge, MA,
USA).

Javed E., Thangavel C., Frara N., Singh J., Mohanty I., Hypolite J., Birbe R., Braverman A.S., Den R.B., Rattan S., Zderic S.A., Deshpande D.A., Penn R.B., Ruggieri MR S.r., Chacko S, & Boopathi E. (2019). Increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(2), 2126-2146.

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Protein Expression Analysis by Western Blotting

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Protein was extracted and analyzed by western blotting as described
previously (10 (link)). Briefly, total proteins
from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride
membranes (Millipore, Bedford, MA). The proteins were subjected to immunoblot
analysis using antibodies specific for desmin, SMA, SMHC, SM22 (Abcam,
Cambridge, MA, USA), vimentin, GFP, JNK1, JNK2, phospho-JNK (Cell Signaling
Technology, Danvers, MA, USA), phospho-myosin light chain (Thr18/Ser19) (Cell
Signaling Technology, Danvers, MA, USA), myosin light chain (Abcam, Cambridge,
MA, USA) and GAPDH (Millipore, Burlington MA, USA) antibodies. The
immunoreactive proteins were visualized as described previously (10 (link)). Equal loading was confirmed by probing the
membranes with anti-GAPDH antibody. Bands were quantified by densitometry using
an Alpha Innotech FluroChem 8800 Image system (Protein Simple, San Jose, CA,
USA). Expression of protein of interest except pMLC₂₀ was normalized
to the expression of GAPDH. Protein bands from immunoblots for pMLC₂₀were quantitated and normalized the data with MLC₂₀.

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Liver Protein Analysis by Western Blot

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Medial lobe liver sections were snap frozen at the time of euthanasia. Liver tissue was homogenized in RIPA buffer containing a protease and phosphatase cocktail (Thermofisher, Waltham, MA). Samples were then prepared with laemmli sample buffer and β-mercaptoethanol at a 4 µg/µl concentration and 32 µg of total protein run down each lane of a 4–20% gradient gel (Biorad, Hercules, California) at 70 V. Samples were then transferred to 0.45 µm nitrocellulose membranes, dried, reconstituted with _ddiH₂O, blocked with LiCor (Lincoln, Nebraska) TBS blocking buffer, and incubated in primary antibody overnight at 4 °C. Primary antibodies include β-actin (Santa Cruz Biotechnology, Dallas, Texas), phospho myosin light chain (Thr18/Ser19) (Cell Signaling Technologies, Danvers, MA), phospho and total p65 (Cell Signaling Technologies, Danvers, MA), phospho and total ERK 1/2 (Cell Signaling Technologies, Danvers, MA), and catalase (Abcam, Cambridge MA). Membranes were then washed with TBS containing 1% tween (TBST), incubated in Licor near-infrared secondary antibodies for 1 h at room temperature, washed again with TBST, and finally imaged with the LiCor Odyssey Clx. Densitometry analysis was conducted in Image-J (National Institutes of Health, Bethesda, Maryland) with phosphor-signal normalized to total-signal and fold change calculated from control.

Bowdridge E.C., DeVallance E., Garner K.L., Griffith J.A., Schafner K., Seaman M., Engels K.J., Wix K., Batchelor T.P., Goldsmith W.T., Hussain S, & Nurkiewicz T.R. (2022). Nano-titanium dioxide inhalation exposure during gestation drives redox dysregulation and vascular dysfunction across generations. Particle and Fibre Toxicology, 19, 18.

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