Protein 4.1 binds to pICln in transfected cells. SiHa cells were electroporated with a GFP-pICln cDNA or a GFP vector alone. (A) Whole cell lysates (∼10 μg) were immunoblotted with a monoclonal antibody against GFP. Lane 1, GFP-pICln–transfected cells; lane 2, GFP vector DNA-transfected cells; lane 3, untransfected SiHa cells. Immunoblot analysis demonstrated that GFP-pICln was expressed in a substantial amount in transfected SiHa cells (lane 1). (B) Whole cell lysates were immunoprecipitated with a polyclonal anti-P4.1 antibody (anti-P80). The proteins were separated by SDS-PAGE, transferred to a membrane, and probed with a monoclonal antibody against GFP. Lane 4, GFP-pICln–transfected cells; lane 5, GFP vector DNA-transfected cells; lane 6, untransfected SiHa cells; lane 7, a negative control (GFP-pICln–transfected cell lysates immunoprecipitated with anti-P80 antibody and reprobed with an irrelevant anti-Flag monoclonal antibody). We did not add β-mercaptoethanol (a chemical reagent that can break a protein disulfide bond) into the sample buffer; therefore, most Ig molecules remained as intact molecules (upper bands), whereas only some dissociated into Ig heavy chain subunits (lower bands).