Figure 5

14-3-3 proteins bind a phospho-peptide centered on Ser354. A) The sequence of POB1 was synthesized by the SPOT method in an array of 15 residue long peptides on a cellulose membrane. Numbers 1 to12 on the left of the membrane are the rows. Numbers on the right (24 to 280) are the numbers of the spotted peptides, reported in the intensity diagram. The first three intense spots in row 1 contain peptides RRFRsLPAAH, RRHRsAPGVR and RRSRsFPVTF (where 's' represents a phosphorylated serine) that serve as positive controls of 14-3-3 interactions. The peptides No. 25-188 (row 2–8) are 15 mers whose overlapping sequences span the POB1 sequence from Leu181 to the carboxy-terminal Leu521. Rows 9–11 contain 15 mer-peptides whose sequence is centered on each single serine or threonine in the POB1 PRD1 (308–365). The peptides from No.193 to No. 228, in row 9 and the first 12 peptides in row 10, contain a centered phospho-serine or phospho-threonine. Their unphosphorylated counterparts, No. 229 to No. 264, occupy the last twelve spots in rows 10 and the 24 spots in row 11. Row 12 contains positive and negative control peptides. The membrane was incubated with purified GST-14-3-3 sigma and probed with anti-GST antibodies. The control membrane, incubated with GST, was blank. The intensity of the interaction was revealed with a peroxidase coupled secondary antibody. The POB1 peptide showing the strongest signal (black triangle) is peptide number 203, whose sequence 347KTHSRASsLDLNKVF361, is centered on phosphorylated Ser354 and shows a signal intensity of 8504 arbitrary units (a.u.), comparable with the strongest positive controls. Its non-phosphorylated counterpart is spot number 238 (intensity 991 a.u.). The peptide centered around Ser322 is No.196 (when phosphorylated) and shows a signal of 491 a.u., while its unmodified counterpart is No.232, 1400 a.u. A comparable result was obtained by probing the same array with GST-14-3-3 isoform zeta. The file reporting the quantitative analysis is in additional file 5. The graph is shown reporting the peptide number referred to the position on the membrane and the intensity measured. B) 3xFLAG 14-3-3 zeta was co-expressed in HEK293 with GFP or different constructs expressing POB1 full length-GFP wild type or bearing the indicated mutations of Ser322 or/and Ser354 into alanine (A) or aspartate (D). Lane 1 and 2 are negative controls: in lane 1, the GFP empty vector was co-transfected with 14-3-3; in lane 2, POB1-GFP is co-transfected with the 3xFLAG empty vector. Upper blot: cell lysates were immunoprecipitated with anti-FLAG antibodies (for 14-3-3) and the co-immunoprecipitated POB1-GFP was revealed with anti-GFP antibodies. POB1-GFP is recovered only when Ser354 is not mutated. In the two gels below, 5% of the cell extract utilized in the immunoprecipitation was analyzed by gel electrophoresis and probed with anti-GFP (for POB1) antibodies or anti-FLAG (for 14-3-3) to assess POB1, GFP and 14-3-3 in input.