Poly-proline interaction of PFN3 and PFN4. (A) Poly-L-proline (PLP) affinity chromatography. PLP interaction of PFN3 (upper panel) and PFN4 (lower panel) was examined by column chromatography and fractions analysed on SDS gels of which only the 14–20 kDa regions are shown. PFN3 bound to the PLP-column and was eluted by 2 M and 4 M urea (protein bands highlightened by frame), while PFN4 failed to interact with PLP and was washed from the column during initial washing steps (protein bands highlightened by frame). L: cell lysate before column; F: flow-through, W: wash without urea (1–9 indicate fraction numbers); E2: 2 M urea eluate; E4: 4 M urea eluate; E8: 8 M urea eluate; M: low mass ladder, Ps: pre-stained marker. (B) Quantitative β-galactosidase assay for selective pair wise Y2H interaction of PFN2, PFN3, and PFN4 with polyproline-rich VASP and DIAPH3. Diploids containing PFN2 and VASP or PFN2 and Diaph3 showed significant activity, reflecting the ability of PFN2 interact with these proteins. PFN3-containing diploids revealed significant but weaker binding to DIAPH3, and no binding to VASP. PFN4 failed to interact with both proteins. Bars show quantitative β-galactosidase activity [milliunits/(ml × cells)] of colonies grown in SD/-LTHA (Leu- Trp- His- Adenine-) high stringency medium. The red line marks the level of the negative control. * designates significant β-galactosidase activities. Positive control: diploids from SD/-LTHA medium containing p53 (pGBKT7–53) and SV40 large T-antigen (pGADT7-T). Negative control: diploids with HE6/GPR64-C-terminus (pGBKT7-H21-21-1) und SV40 T-antigen (pGADT7-T) showing no interaction.
Behnen et al. BMC Cell Biology 2009 10:34 doi:10.1186/1471-2121-10-34