Fig. 2. Co-immunoprecipitation between amphiphysin 2 and SNX4. (A) 3T3 cells were transfected with SNX4 or control RGS14 in the mammalian expression vector pRK5myc. After 24 hours in culture, cells were submitted to hypotonic lysis and membrane/cytosol fractionation. Immunoprecipitations of the endogenous amphiphysin 2 were performed on cytosolic (C) and membrane (M) fractions. Total extracts and anti-amphiphysin 2 immunoprecipitations (IP) were run on SDS-PAGE gels, transferred to nitrocellulose membranes and blotted with relevant antibodies (WB: anti-myc or anti-Amp2). (B) HeLa cells were co-transfected with Bramp2 in pRK5 and SNX4 or control RGS14 in pRK5myc. After 40 hours in culture, cells were submitted to hypotonic lysis and membrane/cytosol fractionation. Anti-myc immunoprecipitations were performed on cytosolic (C) and membrane (M) fractions. Total extracts and anti-myc immunoprecipitations were run on SDS-PAGE gels, transferred to nitrocellulose membranes and blotted with relevant antibodies (WB: anti-Amp2 or anti-myc). Reactive bands were revealed by ECL.