By Balch W.E., Channing J.D., Hall A.
Small GTPases play a key function in lots of elements of latest telephone biology: keep an eye on of telephone development and differentiation; law of phone adhesion and mobile circulation; the association of the actin cytoskeleton; and the law of intracellular vesicular delivery. This quantity plus its significant other Volumes 255 and 256 hide all biochemical and organic assays at present in use for interpreting the function of small GTPases in those features of mobilephone biology on the molecular level.Volume 257 presents specific protocols to successfully produce, regulate, and assay for the functionality of small GTPases considering vesicular site visitors in the course of the secretory pathway of eukaryotic cells.
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Flier, J. Biol. Chem. 271, 1890 (1996). I¢~L. Gnudi, E. U. Frevert, K, L. Houseknecht, P. Erhardt, and B. B. Kahn, Mol. Endocrinol. 11, 67 (1997). 17 p. Kometiani, J. Li, L. Gnudi, B. B. Kahn, A. Askari, and Z. Xie, J. Biol. Chem. 273, 15249 (1998). [3] RADIATION, EGFR, Ras, AND M A P K INTERACTIONS 35 Radiation Induction o f hnmediaw Prima O, and SecotMao, Activations ~{f EGFR and E R K - M A P K Pathway in EGFR-Antisense Carcimmul Cells The ability of radiation to modulate EGFR and E R K - M A P K activity was investigated in EGFR-antisense cells.
H. Warne, A. Khwaja, B. M. Mar(e, D. Pappin, R Das, M. D. Waterfiekl, A. Ridley. and J. Downward. Cell 89, 457 (1997). N,IETIIODS IN E N Z Y M O L O ( ] Y . 00 [4] Ras ACTIVATION OF PI 3-KINASE ANt) Akt E G F R . l-~l 2,~ I n h i b i t i o n o f E R K - M A P K f u n c t i o n by the M E K 1/2 i n h i b i t o r U 0 1 2 6 d u r i n g irradiation e n h a n c e d r a d i a t i o n - i n d u c e d a p o p t o sis. T h e s e data s u g g e s t that i o n i z i n g radiation m a y exert a s e l f - l i m i t i n g effect on its ability to kill a n d to r e d u c e the proliferation o f t u m o r cell E R K - M A P K activity, w h i c h in turn will lead to b o t h increased proliferation o f t u m o r cells as well as o t h e r c y t o p r o t e c t i v e responses.
5 Ixg each) works well and is routinely used. The amount of plasmid per transfection should always be the same and made to l p~g with empty vector when necessary. Add 6 Ixl of Plus reagent, mix, and let stand at room temperature for 15 min. Add 100 Ixl of DMEM" to which 4 fxl of LipofectAMINE has been added. 1 ml of DMEM* and add 104 p~l of this mix per tube. Mix and let stand for 15 rain. Add the 200 ixl of liposome mix to cells that have been washed once and left with 800 b~l of DMEM '~'. After 3 hr, aspirate off and add 2 ml/well of D M E M - 1 0 % (v/v) fetal calf serum.