Western blot anlaysis of extracts from MCF-7 (human), C2C12 (mouse), and BAEC (bovine) cells, using p190-B RhoGAP Antibody.
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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.
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 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 June 2020
Protocol Id: 10
p190-B RhoGAP Antibody detects endogneous levels of total RhoGAP protein.
Human, Mouse, Rat, Monkey, Bovine
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Lys296 of human p190-B RhoGAP. Antibodies are purified by protein A and peptide affinity chromatography.
Rho family GTPases are key regulators of diverse processes such as cytoskeletal organization, cell growth and differentiation, transcriptional regulation, and cell adhesion/motility. The activities of these proteins are controlled primarily through guanine nucleotide exchange factors (GEFs) that facilitate the exchange of GDP for GTP, promoting the active (GTP-bound) state, and GTPase activating proteins (GAPs) that promote GTP hydrolysis and the inactive (GDP-bound) state (1,2).
The p190 RhoGAP proteins are widely expressed Rho family GAPs. p190-A has been characterized as a tumor suppressor, and research studies have shown that loss or rearrangement of the chromosomal region containing the gene for p190-A is linked to tumor development (3,4). p190-A binds the mitogen-inducible transcription factor TFII-I, sequestering it in the cytoplasm and inhibiting its activity. Phosphorylation of p190-A at Tyr308 reduces its affinity for TFII-I, relieving the inhibition (5). p190-A can also inhibit growth factor-induced gliomas in mice (6) and affect cleavage furrow formation and cytokinesis in cultured cells (7).
Mice lacking p190-B RhoGAP show excessive Rho activation and a reduction in activation of the transcription factor CREB (8). Cells deficient in p190-B display defective adipogenesis (9). There is increasing evidence that p190 undergoes tyrosine phosphorylation, which activates its GAP domain (9-11). Levels of tyrosine phosphorylation are enhanced by Src overexpression (10,11). IGF-I treatment downregulates Rho through phosphorylation and activation of p190-B RhoGAP, thereby enhancing IGF signaling implicated in adipogenesis (9).
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