Mouse anti rac1 antibody

Rac1 activity was measured as previously described [26 ]. Briefly, 200 μg of total cellular protein was incubated with GST-PAK-CRIB fusion protein beads (donated by James E. Casanora of the University of Virginia) captured on MagneGST Glutathione Particles (Promega) for 4 h at 4 °C. The particles were then washed three times with washing buffer containing 4.2 mM Na₂HPO₄, 2 mM KH₂PO₄, 140 mM NaCl and 10 mM KCl (pH 7.2), resuspended in 2 x SDS sample buffer and subjected to western blotting analysis using a mouse anti-Rac1 antibody (BD Biosciences).

Cells seeded on glass coverslips in the presence of 1 μg ml⁻¹ dox for 24 h were fixed in 4% formaldehyde and subjected to Duolink in situ PLA using mouse anti-Rac1 antibody (1:100; BD Biosciences, 610650) and rabbit anti-FLII antibody (1:100; Sigma-Aldrich, HPA007084), the respective Duolink in situ PLA probes (Olink Bioscience, anti-mouse 92004-0100, anti-rabbit 92002-0100) and the Duolink in situ detection reagent kit (Olink Bioscience, 92013-0100, 92014-0100) according to manufacturer's instructions. For CHL1 cells, mouse anti-FLII antibody (1:100; Santa Cruz, sc-21716) and rabbit anti-P-Rex1 antibody (1:100; Sigma-Aldrich, HPA001927) were used for the Duolink in situ PLA analysis. Coverslips were mounted onto slides using ProLong Gold antifade reagent with DAPI stain (Life Technologies, P36935) and images were taken using the low light microscope system (× 100 magnification). Phalloidin and DAPI were used as fluorescence markers against the actin cytoskeleton and the nucleus, respectively. Quantification of the Duolink signal was performed as outlined in Supplementary Methods.