To avoid artefacts that could interfere with Yap8 and Ufd2 interaction, including the presence of Gal4 tags and overexpression of fusion proteins, reciprocal co-immunoprecipitation assays were performed using BY4742 cells carrying YAP8-c-myc and UFD2-HA in the centromeric (CEN-ARS) vectors and under the control of native promoters.
The slides were coverslipped with Vectashield (Vector) and examined under an epifluorescence microscope.
129/Sv ES cells were electroporated with 20 µg of linearized targeting vector and selected under G418 treatment.
The slides were mounted with vectashield mounting media (Vector) and viewed under an Olympus Fluoview FV1000 confocal fluorescence microscope.
Fluorescently labelled embryos were mounted in Vectashield (Vector) and examined under a Nikon D-Eclipse C1 confocal scanning unit, mounted on a Nikon Eclipse 90i microscope, using the EZ-C1 3.60 software and a 60x/1.40 NA Apo VC immersion-oil objective.
All putative nvfp cDNAs (except nvfp6 which is very similar to nvfp3) were cloned into pMT/V5-His B vector and expressed under the control of the metallothionein promoter in Drosophila S2 tissue culture cells.
The next day, the slides were incubated for 2 hrs with appropriate secondary antibodies conjugated to either rhodamine (1∶1500, Alexa 594, Molecular Probes) or FITC (1∶500 700, Alexa 488, Molecular Probes) and, after several rinses in PBS, coverslipped with Vectashield or Vectashield with DAPI (Vector) and examined under epifluorescence using an Olympus BX60 microscope.
After estimating DNA concentrations, 120 200 ng were ligated to pIndigoBAC-5 vector and incubated under temperature-cycling conditions.
Sections were rinsed in PBS and in Tris buffer (pH 7.6), mounted with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector), and observed under the AxioImager M2 fluorescence microscope (Carl Zeiss, Germany).
An additional loxP site was inserted into the SacI site downstream of exon V. ES cells were electroporated with a NotI linearized target vector and grown under double selection as previously described [ 33].
