Fig. 3. Crosslinking and co-immunoprecipitation of RseA and YaeL. For lanes 1–8, membrane vesicles carrying indicated combinations of YaeL(H22F)-His6-Myc (shown as PDZ+), YaeLΔPDZ(D402N)-His6-Myc (shown as ΔPDZ), HA-RseA (shown as WT) and HA-RseA165 (shown as 165), were treated with or without DSP. For lanes 9–16, YaeL and RseA were present in separate membrane vesicles (MV1 and MV2), which were mixed and DSP-treated or mock-treated. Membrane proteins were solubilized with 1% N-dodecyl-β-d-maltoside and subjected to immunoprecipitation using immobilized mouse-monoclonal anti-HA antibodies. Proteins recovered were solubilized in SDS, reduced with β-mercaptoethanol to cleave the crosslinkages, and analyzed by SDS–PAGE/immunoblotting using anti-Myc (upper panel) or anti-HA (lower panel) rabbit antibodies. Open arrow, closed arrow, open arrowhead and closed arrowhead indicate YaeL(H22F)-His6-Myc, YaeLΔPDZ(D402N)-His6-Myc, HA-RseA and HA-RseA165, respectively. Direct SDS–PAGE and immunoblotting (without the HA immunoprecipitaiton) demonstrated that all the samples used above contained similar amounts of the YaeL derivatives (not shown).