Western blot analysis of extracts from serum-starved 293T cells treated with resveratrol (50 μM, 1 hr), transfected with a GST-tagged cDNA expression construct encoding either full-length human wild-type SREBP-1c (GST-hSREBP-1c) or full-length mutant SREBP-1c (GST-hSREBP-1c (S372A)), using Phospho-SREBP-1c (Ser372) Antibody (upper) or GST (91G1) Rabbit mAb #2625 (lower). Both expression vectors were generated in Dr. Mengwei Zang's laboratory at Boston University School of Medicine.Learn more about how we get our images
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-SREBP-1c (Ser372) Antibody recognizes transfected levels of SREBP-1c protein only when phosphorylated at Ser372.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser372 of human SREBP-1c protein. Antibodies are purified by protein A and peptide affinity chromatography.
Sterol regulatory element–binding proteins (SREBPs) are basic helix-loop-helix–leucine zipper transcription factors (1,2). Inactive precursor forms of SREBPs are bound to endoplasmic reticulum (ER) membranes (1,2). When cells are starved for cholesterol, SREBPs move from the ER to the Golgi apparatus with the help of SREBP cleavage–activating protein (SCAP) (1,2). In the Golgi apparatus, precursor SREBPs are then cleaved by two proteases, Site-1 protease (S1P) and Site-2 protease (S2P) (1,2). The released N-terminal domains enter the nucleus and bind to sterol response elements in the promoters of a variety of genes responsible for the synthesis of cholesterol (1,2). SREBPs also activate the expression of genes involved in the synthesis of fatty acids and lipids (1,2). Among the isoforms of SREBPs, SREBP-1c activates all lipogenic genes in the liver (3). SREBP-1c has been implicated to contribute to the development of hepatic steatosis in the rodent model of insulin resistance and obesity (3). Recent studies have shown that AMPK interacts with and directly phosphorylates SREBP-1c and SREBP-2 (4). Phosphorylation of SREBP-1c at Ser372 by AMPK, which is stimulated by polyphenols and metformin, inhibits the proteolytic cleavage of SREBP-1c and therefore suppresses the expression of its target genes in the liver (4). This process leads to the reduction of lipid synthesis and accumulation in the liver (4).
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