I found that RGL3 activates Rac1 and Cdc42 and enhance cell migration. I want to prove that RGL3 also activates Arp2/3 complex. Which experiments should I do to prove it?
I do the experiment like this: Cells were co-expressed with RGL3 and Arp3, then was treated with Wiskotatin (Cdc42 inhibitor) or CK666 (Arp2/3 inhibitor). I found the similar disruption effect in RGL3 and ARP3.
Is there any other experiments I can do to prove that RGL3 can activate Arp2/3 complex?
You can do actin polymerization assays, but this requires a lot of purified proteins.
There is a FRET biosensor for Arp2/3, which could be most direct way to measure its activation http://www.sciencedirect.com/science/article/pii/S1097276504005532 Hope it help! Would be glad to help you with experimental setup if you need it. Good luck!
You can co-transfect dominant negative mutants of Rac1 and especially Cdc42 (means using S17N mutations) and measure both the GTP levels of both Rac1 and Cdc42, and Arp2/3 activation. These mutants have low affinity for GDP/GTP and thus undergo in principle a tight complex with their cognate GEFs, e.g. RGL3, and sequesterate them from activating endogenous Rac1 and Cdc42, respectively!!
Thanks Alexis, I am doing the live cell imaging with my protein and GFP-Arp3 ~