Rac1 g lisa kit

To assess Rac1 activation Rac1 G-LISA kit (Cytoskeleton) was used according to the manufacturer’s protocol. Cells were cultured first for 12 h in minimal essential medium (MEM) (Gibco) containing 5% fetal bovine serum (FBS, Thermo Fisher Scientific) after which they were washed with PBS and cultured for an additional 12 h in MEM without FBS. Then cells were washed with PBS and cultured for an additional 4 h in MEM without FBS with or without adding Rac1 inhibitor ZINC69391 to final concentration of 100 µM. Then cells were washed with PBS and incubated for 5 min with EGF (Peprotech) at concentration 100 ng/ml. Protein was isolated using standard G-LISA buffer GL36 (Tris pH 7.5, MgCl₂, NaCl, IGEPAL and SDS). Obtained lysate was aliquoted and frozen in liquid nitrogen. The Rac1 G-LISA kit (Cytoskeleton) contains a Rac-GTP-binding protein linked to the wells of a 96 well plate. Active, GTP-bound Rac1 in cell/tissue lysates will bind to the wells while inactive GDP-bound Rac1 is removed during washing steps. The bound active Rac1 is detected with a Rac1 specific antibody. The degree of Rac1 activation is determined by comparing readings from activated lysates versus non-activated lysates. Plates were read in a Multilabel Counter 1420 VICTOR3V (Perkin Elmer, Waltham, Massachusetts, USA) at 450 nm.

Commercial Rac-1 G-LISA™ kit customized to capture and quantify Rac1-GTP was purchased from Cytoskeleton, Inc (Denver, CO, USA). Rac1-GTP activity was assessed on treated and untreated cell lysates according to the manufacturer’s instructions.