Fig. 3. Determination of the binding site of PKB to periplakin. (A) COS-7 cells were transiently transfected with myc-tagged c-ppl in combination with HA-tagged PKB constructs as indicated. HA-PKB was immunoprecipitated with anti-HA and analysed by western blotting for myc-c-ppl binding by immunoblotting with anti-myc (9E10) (upper panel). Expression of HA-PKB constructs and myc-c-ppl was checked by immunoblotting with anti-HA and anti-myc, respectively. (B) The outcome of the interaction studies by co-immunoprecipitation and yeast two-hybrid analysis. nd, not done. (C) Primary structure of the PH domain of PKB. The positions of the ß-sheets and -helix are indicated by arrows on top and were determined by sequence alignment with other PH domains (see also Ferguson et al., 2000). The nine amino acid deletion that results in loss of periplakin binding is indicated by a black line below the sequence. The conserved W99 residue is depicted in bold and indicated by an arrow.