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Fig. 2. PE treatment causes a gel shift of the GFP-AFAP-110 protein band in western blot analysis that is dependent upon Ser277. (A) Western blot analysis of GFP-AFAP-110 transfected COS-7 cell lysates. 48 hours post transfection cells were either left untreated (–) or treated for 1 hour with 100 nM PMA (+), directly lysed in SDS sample buffer and subjected to SDS-PAGE and subsequent western blotting with GFP antibody. PMA treatment causes a gel shift of the GFP-AFAP-110 protein band. The horizontal line is shown to contrast the gel shift. (B) Protein sequence alignment of the Ser/Thr rich region between different species. Conserved Ser residues that represent putative PKC target sites – [R/K]XX[S/T] and [R/K]X[S/T] (Pearson and Kemp, 1991) – and are underlined and have subsequently been mutated to Ala. (C,D) Western blot analysis of GFP-AFAP-110 phosphorylation mutants. (C) Ser277, Ser278, Ser282 and Ser283 (as indicated in 2B) were mutated to Ala to create the 4A mutant (lanes 1 and 2). For double mutants Ser277 and Ser278 or Ser282 and Ser283 were mutated to Ala to create S277A-S278A or S282A-S283A mutants (277A278A and 282A283A lanes 5,6 and 7,8, respectively). (D) For single mutants, Ser277 or Ser278 were mutated to Ala to create S277A or S278A (277A or 278A, respectively). All constructs were transfected into COS-7 cells and 48 hours post transfection treated as indicated with PMA for 1 hour, while inhibition of PKC was accomplished using the PKC inhibitor GF109203X (GF) for 30 minutes. All blots were probed with anti-GFP antibody and the gelshifted protein bands are indicated by an asterisk. Molecular mass markers are indicated on the left. The horizontal line is shown to contrast the gel shift.
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