Figure 1.
Ubiquitinated FcɛRI and Nedd4 are present within DRMs in RBL cells. RBL cells were transfected with an HA-tagged ubiquitin chimera. (A) Cellular extracts from sensitized RBL cells activated with DNP-BSA for less than 5 min at room temperature and treated (lane 1) or not (lane 2) with MeCD were analyzed by Western blotting. Anti-HA antibody was used to detect HA-tagged ubiquitinated proteins. Arrows indicate where β and γ subunits of the FcɛRI migrate. (B) Sensitized RBL cells were activated (+Ag) or not (−Ag) with DNP-BSA for less than 5 min at room temperature before lysis and analysis by SDS/PAGE and Western blotting. After floatation of Triton X-100 cell extracts, proteins were precipitated by using MeOH/CHCl3 followed by an acetone wash. The analysis was carried out by SDS/PAGE and Western blotting using an anti-HA antibody. Arrows indicate where β and γ subunits of the FcɛRI migrate. (C) Filters presented in B were reprobed with anti-Lyn (C), anti-Cbl (D), and anti-Nedd4 (E) antibodies.