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. 2002 Dec;13(12):4167–4178. doi: 10.1091/mbc.02-05-0068

Figure 4.

Figure 4

Nonpolymerizable actin mutants inhibit SRF activation. (A) Actins G13R and R62D block serum-induced SRF activation. NIH3T3 cells were transfected with 0.2, 0.5, 1.0, or 2.0 μg of expression plasmid encoding FLAG-tagged wild-type actin, actin G13R, actin R62D, or empty vector, together with the SRF reporter 3D.ALuc. After serum stimulation, luciferase activities were measured and expressed relative to activation by SRF-VP16, taken as 100. Inset shows a FLAG immunoblot of cell extracts. Data show mean of at least three independent experiments with SEM indicated by the bars. (B) Actin/VP-16 fusion proteins inhibit SRF activation. NIH3T3 cells were transfected with expression plasmids encoding FLAG-tagged wild-type actin, NLS-actin, actin VP.N, actin NLS-VP.N (0.5 μg each), or actin VP.C (2 μg), or empty vector, together with the SRF reporter gene 3D.ALuc, and reporter activity was analyzed as in A. (C) CCT-binding mutant actin G150P does not inhibit SRF activation. Cells were transfected with the indicated mutants and 3D.ALuc as in A, except that activities were normalized to a cotransfected tk-luc reference plasmid rather than total recovered protein to minimize variations arising from the toxicity of actin G150P. (D) Nonpolymerizable actins inhibit SRF activation by cytochalasin D. Cells were transfected with actin expression plasmids (0.5, 1.0, or 2.0 μg) and 3D.ALuc and analyzed as in C, with stimulation by 10 μM cytochalasin D before analysis of reporter gene activity.