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. 2001 Dec;21(23):8225–8235. doi: 10.1128/MCB.21.23.8225-8235.2001

FIG. 2.

FIG. 2

The Ras-Ral-induced phosphorylation sites in AFX. (A) Deletion mutants of AFX. Abbreviations: DB, DNA binding domain; TA, transactivating domain. (B) 32P-labeled pMT2-HA-AFX-WT, pMT2-HA-AFX-1–416, pMT2-HA-AFX-Δ419–443, pMT2-HA-AFX-Δ444–461, or pMT2-HA-AFX-Δ462–501 AFX was isolated from A14 cells which were cotransfected with p-SVE-RasV12 and analyzed as described in Fig. 1A. The same results were obtained when cells were cotransfected with Rlf-CAAX (data not shown). (C) 32P-labeled pMT2-HA-AFX-T447A, pMT2-HA-AFX-T451A, pMT2-HA-AFX-T454A, or pMT2-HA-AFX-T447A/T451A was isolated from A14 cells which were cotransfected with p-SVE-RasV12 and analyzed as described in Fig. 1A. The same results were obtained when cells were cotransfected with Rlf-CAAX (data not shown). (D) A14 cells were cotransfected with either 2 μg of pCMV-GFP-Pep17-WT, pCMV-GFP-Pep17-T447S, pCMV-GFP-Pep17-T451S, or pCMV-GFP-Pep17-T447S/T451S in combination with 2 μg of p-SVE-RasV12 (or pcDNA3-Myc-Rlf-CAAX [data not shown]). Following exposure to film, bands were cut out of the blot and processed for phosphoamino acid analysis. Positions of phosphoamino acids are as indicated.