By Balch W.E., Channing J.D., Hall A.

Small GTPases play a key position in lots of facets of up to date cellphone biology: keep an eye on of phone development and differentiation; rules of telephone adhesion and mobile circulation; the association of the actin cytoskeleton; and the legislation of intracellular vesicular delivery. This quantity plus its spouse Volumes 255 and 256 conceal all biochemical and organic assays at the moment in use for interpreting the function of small GTPases in those elements of phone biology on the molecular level.Volume 257 offers particular protocols to successfully produce, alter, and assay for the functionality of small GTPases excited about vesicular site visitors during the secretory pathway of eukaryotic cells.

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Small GTPases and Their Regulators Part G

Small GTPases play a key position in lots of elements of up to date phone biology: regulate of cellphone progress and differentiation; legislation of telephone adhesion and telephone move; the association of the actin cytoskeleton; and the rules of intracellular vesicular delivery. This quantity plus its spouse Volumes 255 and 256 conceal all biochemical and organic assays at present in use for studying the function of small GTPases in those elements of telephone biology on the molecular point.

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Dictcrle. A. Pavil-ani,and M. Mehtali. ,/. Viro/. 70, 4805 1996). 2o U. Kasid, S. Suy. t{ l)ent, T. Whilcside, and T. W. Sturgill, Natztre (Lot;do,) 382, 316 (I 996). Minutes after irradiation 0 1 2 5 30 60 120 180240300 2 Gy EGFR-Y tied 0 qDmqDeieieim 4t- E G F R total protein blot Minutes after irradiation I 2 5 30 60 120180 240 300 4~ 2 Gy+ asEGFR EGFR-Y E G F R total protein blot --e-- 2Gy 2 Gy+ 2 FM U0126 - - I - - 2 Gy + RasN17 2 Gy + EGFR-antisense ~4 a. < 3 r. o : 2 o It, 0 S0 100 150 200 250 300 Time (min) FIG.

Pullen, R B. Dennis, M. Andjelkovic, A. Dufner, S. C. Kozma, B. A. Hemmings,and G. Thomas, Science 279, 707 (1998). 18C. Sutherland, D. G. Campbell, and E Cohen, Ear. J. Biochem. 212, 581 (1993). 19T. W. Sturgill, L. B. Ray, E. Erikson, and J. L. Mallet, Nature (London) 334, 715 (1988). 20 T. L. Fisher and 1. Blenis, Mol. Cell. Biol. 16, 1212 (1996). 21 K. N. Dalby, N. Mortice, F. B. Caudwell, J. Avruch, and R Cohen, J. Biol. Chem. 273, 1496 (1998). [5] p70 S0 KINASE AND R S K ASSAYS 47 immunoblotting, and recognize R S K I of avian, murine, rat, and human origin; only data for anti-RSK(6) are shown below.

Transfer and combine the lower phases of tubes 1 and 2 in a clean tube (tube 3). Samples can be stored at - 2 0 :~ at this stage. Dry in a Speed-Vac vacuum concentrator. Thin-Layer ChromatographyAnalysis The large increase in PIP3 seen in COS cells transfected with p110 and Ras can be easily seen on a TLC plate as discussed above. If HPLC analysis is going to be performed this step is optional. If a 6-cm dish has been used, one-tenth of the chloroform phase is dried separately and used for analysis by TLC.

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