Figure 5.

 In vivo binding of NPF-containing proteins to Eps15. (A) Coimmunoprecipitation of Eps15 with endogenously expressed NUMB and RAB. Total cellular proteins from NIH–3T3 cells were obtained under mild lysis conditions to preserve protein–protein interactions, and 5 mg of protein was immunoprecipitated with an anti-RAB (Ip RAB), an anti-NUMB (Ip NUMB), or a control serum (C). After SDS–PAGE, coimmunoprecipitating Eps15 was detected by immunoblot with an anti-Eps15 antibody. (B) Eps15 coimmunoprecipitation with HA-tagged RAB and NUMB proteins. (Top) C33A cells were transfected with expression vectors engineered to express HA-tagged RAB or NUMB proteins (HA–RAB or HA–NUMB lanes, respectively) or mock transfected (− lanes). Total cellular lysates (100 μg) were immunoblotted with an anti-HA antibody. (Bottom) Five milligrams of total cellular proteins from HA–RAB or HA–NUMB transfectants (HA–RAB Tfx and HA–NUMB Tfx, respectively) obtained as in A were immunoprecipitated with the anti-HA antibody (HA lanes) or with a control serum (C lanes) and detected by immunoblot with an anti-eps15 antibody. (C) Eps15 coimmunoprecipitation with HA-tagged NUMB and NUMB mutant. C33A cells were transfected with expression vectors encoding HA-tagged NUMB or an HA-tagged NUMB mutant in which the NPF motif was mutagenized to NAA (lanes HA–NUMB and HA–NUMB/NAA, respectively), or mock transfected (− lane). (Top) Total cellular lysates (100 μg) were immunoblotted with an anti-HA antibody; (middle) 5 mg of total cellular proteins obtained as in A was immunoprecipitated with an anti-HA antibody and detected by immunoblot with an anti-Eps15 serum; (bottom) total cellular lysates (100 μg) were immunoblotted with an anti-Eps15 antibody. Lanes marked LYS in A–C were loaded with 50 μg of total cellular proteins to serve as a reference for the position of Eps15. The positions of Eps15, NUMB, and RAB are also indicated.