Full Name
srGAP2 symmetric dimethylated R297
Immuno Sequence
CKTATAGR*SKSFNNHRP [residues 921 - 936 of human]
Mono/Poly clonal
Polyclonal
Gel Image
Figure 1 Peptide dot blots The indicated amounts (0.05 to 1.0 μg) of synthetic dimethylated (M) and phosphorylated (P) peptides, and their unmodified (C) counterparts were spotted onto nitrocellulose. The peptides were immunoblotted with 0.1 μg/ml of purified antibody preincubated in the presence or absence of dimethylated immunogen or the unmodified peptide (10 μg/ml). The immunogen was symmetrically dimethylated Arg927 on srGAP2 (CKTATAG(R*)SKSFNNHRP, residues 921-926 of human srGAP2)
HEK293 cells were transfected to express GFP-tagged forms of srGAP2 and point mutants Arg927Ala and Arg927Lys. Cells were serum starved for 5 h and stimulated as indicated with PMA (100 ng/ml) for 30 min +/- Gö6983 (1 μM) for 30 min. Cell lysates (40 μg) were blotted for Erk1/2, pErk1/2, pSer930 (of srGAP2) and GFP. GFP-Trap was used to isolate the srGAP2 proteins, which were Western blotted with the SDMA-R927-srGAP2 antibody (S271D), and also with an antibody from AbCam that recognizes symmetrical and non-symmetrical dimethylated arginine and mono-methylated arginine (SDMA/ADMA/MMA). Blots were also performed to detect phospho-Ser930 of srGAP2 and GFP. 14-3-3 binding assays were performed by both a Far-Western approach and detection of co-precipitated endogenous 14-3-3 (K-19, pan-14-3-3 antibody). This experiment shows that the recognition of srGAP2 by the S271D antibody is dependent on Arg927 of srGAP2, this does not allow us to say whether the antibody is selective for Arg927 that is dimethylated.
Effect of PRMT inhibition on recognition of srGAP2 by S271D antibody HEK293 cells were transiently transfected to express GFP-tagged srGAP2. Cells were serum starved for 5 h (in 0.5% FCS) and then treated with AMI-1 (50 μM) for 1 h or 24 h and stimulated or not with PMA (100 ng/ml) for 30 min +/- Gö6983 (1 μM) for 30 min. Cell lysates (40 μg) were blotted with the S271D antibody raised against symmetrically dimethylated Arg927 of srGAP2, and with the Abcam pan-methylation (SDMA/ADMA/MMA) antibody). AMI-1 is an inhibitor of methyltransferases, including PRMT5. This experiment gives an indication that the AMI-1 might decrease the S271D antibody signal, but is not conclusive, and further work is needed.
Sheep No
S271D
Unit Source
Aliquot
0.1mg
Price
£150.00