Fig. 4. The CAAX motif of C630 is required for delaying progression through G2/M. HeLa cells were infected with a control retrovirus, retroviruses encoding myc-tagged C630 and the four CAAX mutants, C:S, V:D, 4 and 150. Puromycin-resistant cells were analysed to determine protein expression, localisation, relative growth rates and cell-cycle timing. (A) Growth curves showing that the CAAX mutants induce a less severe growth defect relative to C630. (B) DNA content histograms of asynchronous cultures showing that the CAAX mutants induce a less profound G2/M accumulation relative to C630. (C) Graph plotting the 4n/2n DNA content ratio, as determined by DNA content flow cytometry, following release from a G1/S block. Unlike C630, the CAAX mutants do not induce a significant G2/M delay. (D) Immunoblot showing the relative expression levels of C630 and the CAAX mutants. The arrow shows the position of C630 while the asterisk indicates a nonspecific background band that serves as a convenient loading control. (E) Immunofluorescence analysis 12 hours after release from a G1/S block showing that the C630 CAAX mutants localise to the nucleus during interphase.

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