Figure 2
In vitro interaction of PFN3 and PFN4 with actin. A) Yeast two-hybrid (Y2H) interaction assays of PFN2, PFN3, and PFN4 with β-actin (left panel). Plate growth assays were performed on minimal media in the absence of histidine (SD/-LTH) or adenine (SD/-LTA) or both (SD/-LTHA). Interaction is indicated by growth of diploid colonies. Upper panel shows assay for profilin-pGADT7 × actin-pGBKT7 on SD/-LTHA medium. Lower panel shows positive control (actin-pGBKT7 × huProfilin-2-pGADT7) on the left;, negative control (actin-pGBKT7 × pGADT7) on the right. Quantification was carried out by β-galactosidase assay (right panel) using diploids from SD/-LTHA-medium Red line marks the negative value; * designates significant activities. Positive control: diploids 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). B) Western blot analysis of hemagglutinin epitope (HA)-tagged β-actin co-immunoprecipitated with c-myc-tagged PFN2, PFN3, and PFN4. Analyses employing the c-myc- antibody showed that the three profilin isoforms (~14 kDa) were precipitated (upper panel; highlightened by arrow). Analyses employing the anti-HA- antibody showed that actin-HA (~45 kDa) was co-precipitated with PFN2 and PFN3, but not with PFN4 (bottom panel; highlightened by arrow). The ~28 kDa and ~50 kDa protein bands resulted from the light and heavy chains of the c-myc-antibody.