FAC binds to GRP94 in vivo in transfected cells. (A) COS-1 cells transfected with the indicated constructs (vector, pED6; remainder as described in the Materials and Methods) were processed after 48 hours, and cytoplasmic extracts were prepared in a final concentration of 50 mmol/L NaCl. Extracts were immunoprecipitated with either anti-FAC antiserum and protein A-agarose sequentially (+) or with protein A-agarose (−) only, boiled, resolved by SDS-PAGE, and probed for GRP94 expression by immunoblotting. (B) Analysis of transfected COS-1 cell lysates directly by immunoblotting and probing with anti-GRP94 antibody. The endogenous COS-1 GRP94 appears slightly smaller than the transfected human GRP94 cDNA (shown as bent arrows).