I am trying to determine if the Arp2/3 complex is important in my system. I was wondering if anybody could recommend a good control to make sure the CK-666 is effective in inhibiting the ARP2/3 complex in these conditions by immunofluorescence?
Thank you very much!
Guada
Hi Maria,
I am assuming that you want to look at the levels of filamentous actin and/or actin polymerization. If you inhibit Arp2/3 I would expect actin depolymerization which you could check in IF simply by a phalloidin staining which recognizes F-Actin. As an additional control you could use drugs that directly act on actin such as Latrunculin A which will immediately depolymerize F-Actin or Jasplakinolide which will stabilize F-Actin.
Hope that helps.
Cell spreading assay could visualize and quantify the effect of Actin polymerization. For non-muscle cells such as fibroblasts adhere and spread after being introduced to cell culture substrata. An inhibition of non-muscle myosin by drugs (various good ones available) increases rate of cell spreading (J Cell Sci. 2003 Apr 15;116(Pt 8):1617-25.). Under this condition, the cell spreading depends mainly on actin polymerization. Thus, I expect to the effect of Arp2/3 inhibitor should be clear. Cell area analysis can quantify these effects.
As suggested, when G-actins are sequestered by drugs (Latrunculin), spreading should also be inhibited (potential control). Access to GFP-Arp or GFP-Capping protein (J Cell Biol. 1998 Dec 28;143(7):1919-30.) may also help to analyze drug effects. Depending on your experimental questions, control selections may need to be adjusted.
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