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The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity.

Butler J, Smyth N, Broadbridge R, Council CE, Lee AG, Stocker CJ, Hislop DC, Arch JR, Cawthorne MA, Malcolm East J - Arch. Biochem. Biophys. (2015)

Bottom Line: The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump.Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA.In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK.

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(A) Characterisation of sarcolipin levels in mouse atria and rabbit skeletal muscle by semi-quantitative western blotting. 30 μg (protein) of homogenised pooled left and right atrial tissue from 8 to 10 week old FVBN mice was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 5.0 and 2.5 ng of peptide were included as standards (lanes 1, 2 and 3 respectively). Following the transfer of the proteins from the gel to PVDF membranes the blots were probed with anti-sarcolipin antibody, followed by a goat anti-rabbit fluorophore conjugated antibody. (B) SR, 1 μg, from a 3 kg New Zealand white rabbit was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 40 and 60 ng (lanes 1–3). The blots were visualised and analysed using the LI-COR ODYSSEY detection system. Blots shown are typical of at least two determinations.
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f0005: (A) Characterisation of sarcolipin levels in mouse atria and rabbit skeletal muscle by semi-quantitative western blotting. 30 μg (protein) of homogenised pooled left and right atrial tissue from 8 to 10 week old FVBN mice was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 5.0 and 2.5 ng of peptide were included as standards (lanes 1, 2 and 3 respectively). Following the transfer of the proteins from the gel to PVDF membranes the blots were probed with anti-sarcolipin antibody, followed by a goat anti-rabbit fluorophore conjugated antibody. (B) SR, 1 μg, from a 3 kg New Zealand white rabbit was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 40 and 60 ng (lanes 1–3). The blots were visualised and analysed using the LI-COR ODYSSEY detection system. Blots shown are typical of at least two determinations.

Mentions: The semi-quantitative western blotting method using synthetic mouse sarcolipin was validated by estimating the levels of sarcolipin in the atria of FVBN mice. Fig. 1A is a western blot showing a comparison of the content of sarcolipin in 30 μg of mouse atria in lane 4 with 25, 5.0 and 2.5 ng of synthetic sarcolipin (lanes 1–3 respectively). Sarcolipin migrates by SDS–PAGE with an apparent Mr of 4000. The amount of sarcolipin in 30 μg of atrial tissue is approximately 9 ng. Using the estimate of SERCA obtained by western blotting of 14 μg/mg atrial tissue (data not shown), which is comparable to the value of 10 μg SERCA/mg atrial tissue obtained by [7]), this gives a molar ratio of 0.50 mol sarcolipin/mol SERCA. This value is similar to that previously reported: 1.24 mol sarcolipin/mol SERCA [7]. Similarly the blot shown in Fig. 1B compares 1 μg of rabbit total skeletal muscle SR (lane 4) with 20, 40 and 60 ng synthetic mouse sarcolipin (lanes 1–3 respectively). One microgram of SR contains approximately 27 ng sarcolipin. These data give a value of 1.2 mol sarcolipin/mol SERCA, assuming that rabbit muscle SR contains 70% SERCA (estimated from SDS–PAGE of SR stained with SYPRO orange). This is similar to the values previously reported for rabbit soleus and extensor digitorum longus muscles of 0.87 and 0.37 mol sarcolipin/mol SERCA respectively [7]. Even though the sequences of rabbit and mouse sarcolipin are different, the use of an antibody against the mouse sarcolipin sequence to quantify the rabbit homologue is justified because the antibody is raised against the peptide VRSYQY, corresponding to the C-terminal sequence of sarcolipin from numerous mammalian species, including mouse, rabbit and human.


The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity.

Butler J, Smyth N, Broadbridge R, Council CE, Lee AG, Stocker CJ, Hislop DC, Arch JR, Cawthorne MA, Malcolm East J - Arch. Biochem. Biophys. (2015)

(A) Characterisation of sarcolipin levels in mouse atria and rabbit skeletal muscle by semi-quantitative western blotting. 30 μg (protein) of homogenised pooled left and right atrial tissue from 8 to 10 week old FVBN mice was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 5.0 and 2.5 ng of peptide were included as standards (lanes 1, 2 and 3 respectively). Following the transfer of the proteins from the gel to PVDF membranes the blots were probed with anti-sarcolipin antibody, followed by a goat anti-rabbit fluorophore conjugated antibody. (B) SR, 1 μg, from a 3 kg New Zealand white rabbit was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 40 and 60 ng (lanes 1–3). The blots were visualised and analysed using the LI-COR ODYSSEY detection system. Blots shown are typical of at least two determinations.
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Related In: Results  -  Collection

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f0005: (A) Characterisation of sarcolipin levels in mouse atria and rabbit skeletal muscle by semi-quantitative western blotting. 30 μg (protein) of homogenised pooled left and right atrial tissue from 8 to 10 week old FVBN mice was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 5.0 and 2.5 ng of peptide were included as standards (lanes 1, 2 and 3 respectively). Following the transfer of the proteins from the gel to PVDF membranes the blots were probed with anti-sarcolipin antibody, followed by a goat anti-rabbit fluorophore conjugated antibody. (B) SR, 1 μg, from a 3 kg New Zealand white rabbit was separated by SDS–PAGE (lane 4) and synthetic sarcolipin, 20, 40 and 60 ng (lanes 1–3). The blots were visualised and analysed using the LI-COR ODYSSEY detection system. Blots shown are typical of at least two determinations.
Mentions: The semi-quantitative western blotting method using synthetic mouse sarcolipin was validated by estimating the levels of sarcolipin in the atria of FVBN mice. Fig. 1A is a western blot showing a comparison of the content of sarcolipin in 30 μg of mouse atria in lane 4 with 25, 5.0 and 2.5 ng of synthetic sarcolipin (lanes 1–3 respectively). Sarcolipin migrates by SDS–PAGE with an apparent Mr of 4000. The amount of sarcolipin in 30 μg of atrial tissue is approximately 9 ng. Using the estimate of SERCA obtained by western blotting of 14 μg/mg atrial tissue (data not shown), which is comparable to the value of 10 μg SERCA/mg atrial tissue obtained by [7]), this gives a molar ratio of 0.50 mol sarcolipin/mol SERCA. This value is similar to that previously reported: 1.24 mol sarcolipin/mol SERCA [7]. Similarly the blot shown in Fig. 1B compares 1 μg of rabbit total skeletal muscle SR (lane 4) with 20, 40 and 60 ng synthetic mouse sarcolipin (lanes 1–3 respectively). One microgram of SR contains approximately 27 ng sarcolipin. These data give a value of 1.2 mol sarcolipin/mol SERCA, assuming that rabbit muscle SR contains 70% SERCA (estimated from SDS–PAGE of SR stained with SYPRO orange). This is similar to the values previously reported for rabbit soleus and extensor digitorum longus muscles of 0.87 and 0.37 mol sarcolipin/mol SERCA respectively [7]. Even though the sequences of rabbit and mouse sarcolipin are different, the use of an antibody against the mouse sarcolipin sequence to quantify the rabbit homologue is justified because the antibody is raised against the peptide VRSYQY, corresponding to the C-terminal sequence of sarcolipin from numerous mammalian species, including mouse, rabbit and human.

Bottom Line: The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump.Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA.In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK.

Show MeSH