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Fig. 1. The intracellular domain of CD21 interacts with FHOS. (a) in yeast. A GAL4 activation domain plasmid encoding the C-terminal amino acids of FHOS was serially diluted (10–1-10–4; UD, undiluted) and co-transformed into a yeast reporter strain with plasmids encoding the GAL4 DNA binding domain fused to one of the following: the 34 aa residues of the CD21 cytoplasmic tail (CD21-CT), the 34 aa residues of the CD21 cytoplasmic domain plus nine residues of the transmembrane domain (CD21-TMCT), lamin, the 105 aa residues of the CAR cytoplasmic domain (CAR-CT), or bacteriophage T7 gene 2 (T7/gp2; see Materials and Methods). Co-transformants were selected on minimal medium. Growth (i.e. activation of the HIS3 reporter) indicates a positive interaction. +C (positive control) represents co-transformation of plasmids encoding SNF1 and SNF4, proteins that interact in the two-hybrid assay. (b) In vitro. Purified fusion proteins expressing the C terminus of FHOS, GST-FHOS, GST alone or GST-TK (irrelevant protein) were incubated with cell lysates prepared from the B-cell lines Raji (CD21+; lanes 2-4) and Nalm6 (CD21–; lanes 5 and 6). Proteins interacting with FHOS were co-purified on glutathione beads, separated by SDS-PAGE and detected by immunoblot using a monospecific rabbit anti-CD21 antiserum. A total protein lysate from Raji cells (lane 1) was analyzed as a CD21 positive control. (c) In vivo. 293T cells were transfected with pG5
CAT-EGFP (reporter) plus the desired combination of bait (pM-X) and target (pVP16-X) vectors as indicated. X denotes the fusion protein of interest. Twenty-four hours after transfection, cells were identified with Hoechst (nuclear stain), fixed, and analyzed by fluorescence microscopy. Fluorescent EGFP+ cells indicates a positive interaction between bait and target fusion proteins. Top panel (method verification): EGFP induction was assessed using positive control vectors (pM53-VP16 and pM-53 + pVP16-T) compared with negative control vectors (pM-53 + pVP16-CP, control protein; Clontech). Middle panel: EGFP induction following co-transfection of recombinant vectors encoding the cytoplasmic domain of CD21, CD21.CT together with the C terminus of FHOS, FHOS.CT. Right and left panels show reciprocal experiments. Bottom panel (control), EGFP induction following transfection of CD21.CT or of FHOS.CT together with cognate two-hybrid vectors encoding irrelevant proteins (T antigen or p53).
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