The Rac activity assay was performed using the Rac-GEF (guanine-nucleotide exchange factors) Assay Kit (Cell Biolabs, San Diego, CA, USA).
We used the Rac small-molecule inhibitor NSC23766 to analyze its effects on NPM ALK-induced iNPM ALK-inducedvinvasivenessALK ALCL cell lines Cost (small-cells vitront) and Karpas 299 (common type).
In order to assess whether or not EspT activates Rac-1 and Cdc42, we performed pull-down assays using the Cdc42 Rac-1 interacting binding domain (CRIB) of Pak, which binds activated Rac-1 and Cdc42 and the CRIB of WASP, which binds only activated Cdc42.
It is however important to keep in mind that even if proteins were isolated using the ABE/Acyl-RAC method of labeling followed by click chemistry, all proteins that contain a thioester bond, not related to S-palmitoylation, will be recovered and thus constitute false positives.
Excluding two-digit postcode matching but allowing matching using the postcode of the RAC facility (instead of that of the person's pre-RAC usual residence) led to similar gains under the SLG strategy (320 additional true links) and significant (but somewhat fewer) gains under the PC strategy (399 additional true links compared with 482 for PC v2).
While previous experiments tested for a role for Rac by microinjection or by overexpression of a constitutively active form of Rac, we activated instead endogenous Rac by using the chemically-induced translocation of a Rac specific guanine nucleotide exchange factor, Tiam1 [8].
In order to confirm the enzyme BVMO activity and to assess its enantioselectivity, reactions were carried out using the model substrate rac-bicyclo[3.2.0]hept-2-en-6-one since both enantioselectivity and regioselectivity, can be simultaneously tested (Mihovilovic 2005).
We measured the activity of Rac1 by means of a pulldown assay using the GST-fused Rac-binding domain of p21 protein-activated kinase (PAK) beads.
Affinity purification assays to measure the levels of endogenous GTP-bound Rac1 and Cdc42 were performed by using the Cdc42- and Rac-binding domain of p21 activated kinase (GST-PAK), as previously described [ 45]. siRNAs specific for Tiam1 or controls (Santa Cruz) were transiently transfected into cells by using SiLentFectTM lipid reagent (Bio-Rad), according to the manufacturer's protocols.
