s1pr1 phosphorylation  (Bioss)

Journal: Skeletal Muscle

Article Title: Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: Administration of S1P leads to increased levels of S1PR1 and P-rpS6 in vivo . ( A ) Experimental schematic of S1P and PBS (vehicle) injected daily for the first 72 hours into TAs of uninjured mdx 4cv mice (n = 4, 2.5-MO, left TAs injected S1P, right TAs injected PBS). ( B ) Western blot analysis of injected TAs (n = 3, 2.5-MO mdx 4cv ) indicates that administration of S1P significantly increases S1PR1 levels. ( C ) Western blot analysis of injected TAs (n = 4, 2.5-MO mdx 4cv ) for total, and P-Akt, P-mTOR and P-rpS6, reveals that total and P-rpS6 were significantly higher with S1P treatment. Increased levels of total and P-rpS6 suggest that S1P administration promotes protein synthesis in mdx muscles. * P <0.05 by student’s t -test. Error bars represent SEM. MO, month-old; P-Akt, phosphorylated Akt; P-mTOR, phosphorylated mammalian target of rapamycin; P-rpS6, phosphorylated ribosomal protein S6; rpS6, ribosomal protein S6; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; SEM, standard error of the mean; TA, tibialis anterior.

Article Snippet: For S1P receptor staining, slides were fixed with 4% formaldehyde for 5 minutes and stained with rabbit polyclonal IgG antibodies against S1PR1, S1PR3 (Cayman Chemical, Ann Arbor, MI, USA) and phosphorylated S1PR1 (raised against Thr236, Assay Biotechnology, Sunnyvale, CA, USA), all applied at a dilution of 1:200 for 2 hours.

Techniques: In Vivo, Injection, Western Blot, Muscles

Journal: Skeletal Muscle

Article Title: Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P <0.005 by student’s t -test. Error bars represent SEM. CTX, cardiotoxin; eMyHC, embryonic myosin heavy chain; IgG, immunoglobulin G; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; S1PR3, S1P receptor 3; SEM, standard error of the mean; TA, tibialis anterior.

Article Snippet: For S1P receptor staining, slides were fixed with 4% formaldehyde for 5 minutes and stained with rabbit polyclonal IgG antibodies against S1PR1, S1PR3 (Cayman Chemical, Ann Arbor, MI, USA) and phosphorylated S1PR1 (raised against Thr236, Assay Biotechnology, Sunnyvale, CA, USA), all applied at a dilution of 1:200 for 2 hours.

Techniques: Injection, Activation Assay, Muscles, Liquid Chromatography with Mass Spectroscopy, Staining, Control, Liquid Chromatography, Mass Spectrometry

Journal: Skeletal Muscle

Article Title: Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: Longer-term treatment with THI elevated muscle force in uninjured mdx EDL muscles. ( A ) Experimental schematic outlining the treatment regimen. Beginning at 4 weeks of age, mdx 4cv mice (1-MO males) were treated for 4 weeks ad libitum with 50 mg/l THI (n = 4) or vehicle (n = 3) in drinking water. ( B ) Myography analysis of EDL muscles reveals a significant increase in maximal specific force with THI treatment. * P <0.05 by student’s t -test. Error bars represent SEM. ( C ) Summary of findings: S1P can act to not only promote myogenic cell activation and muscle repair, but also enhance muscle fiber size and force, possibly through S1PR1 mediated signaling. EDL, extensor digitorum longus; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole.

Article Snippet: For S1P receptor staining, slides were fixed with 4% formaldehyde for 5 minutes and stained with rabbit polyclonal IgG antibodies against S1PR1, S1PR3 (Cayman Chemical, Ann Arbor, MI, USA) and phosphorylated S1PR1 (raised against Thr236, Assay Biotechnology, Sunnyvale, CA, USA), all applied at a dilution of 1:200 for 2 hours.

Techniques: Muscles, Activation Assay