4.1R interacts with eIF3-p44 in vivo.

Cell lysates prepared from Molt-4 cells were immunoprecipitated with preimmune serum (lane 2) or anti-eIF3-p44 antibody (lane 3). Bound protein complexes were analyzed by immunoblotting with antibodies against the N-terminal portion of 135-kd 4.1R (A, anti-N-4.1R) or against the C-terminal 22/24-kd domain of 4.1R (B, anti-C-4.1R antibody). Lane 1 was loaded with Molt-4 cell lysates (10 μg) and immunoblotted with anti-N-4.1R (A), anti-C-4.1R (B), or anti-eIF3-p44 antibody (C). The 135-kd 4.1R isoform was specifically detected by anti-N-4.1R antibody (A, lane 3), whereas 2 alternative splicing isoforms of 4.1R (135-kd and 80-kd) were recognized by anti-C-4.1R antibody (B, lane 3). (C) Reverse immunoprecipitation. Cell lysates prepared from Molt-4 cells were immunoprecipitated with preimmune serum (lane 2) or anti-C-4.1R antibody (lane 3) and analyzed by immunoblotting with anti-eIF3-p44 antibody. Lane 1 represents a positive control in which Molt-4 cell extracts were immunoblotted with anti-eIF3-p44 antibody. Samples were dissolved in SDS sample buffer containing β-mercaptoethanol (A, B) or lacking β-mercaptoethanol (C, lanes 2 and 3).