Fig. 4. HET-C^PA::HA and HET-C^OR::GFP form a heterocomplex in incompatible transformants. (A) Anti-HA antibody immunoprecipitated cellular fractions from CJ44 (het-c^OR::GFP + het-c^OR::HA) compatible and (het-c^OR::GFP + het-c^PA::HA) incompatible transformants and their untagged counterparts were subjected to western analysis using anti-GFP antibodies. (B) The blot in (A) was stripped in stripping buffer (62.5 mM Tris–HCl pH 7.6, 100 mM 2-mercaptoethanol, 5% SDS) at 55°C for 30 min, rinsed in PBS, blocked and re-probed with anti-HA antibody. HET-C homocomplexes were undetectable in CJ44 (het-c^OR::GFP + het-c^OR::HA) transformants (OR+OR tagged). PM, plasma membrane fraction; CT, cytoplasmic fraction; CW, cell wall fraction. (C) HET-C heterocomplex formation was detected in three het-c allelic combinations by co-immunoprecipitation experiments. Plasma membrane fractions were isolated from CJ44 (het-c^PA::HA + het-c^OR::GFP), (het-c^OR::HA + het-c^OR::GFP) and (het-c^OR::HA + het-c^GR::GFP) incompatible transformants and subjected to co-immunoprecipitation, as above. MW markers are indicated.