Figure 2.

The mutant envelope proteins are processed normally in NIH 3T3 cells, with the exception of F607V. Cells (5 × 10⁵) that were stably expressing the various mutant envelope proteins were labeled with 50 μCi/ml ³⁵S-cysteine/[³⁵S]methionine for 4 h before lysis with 1× RIP buffer. The cell debris was removed before two rounds of immunoprecipitation of the envelope proteins with antibody against SU. The immunoprecipitated envelope proteins were analyzed on an 8% SDS-PAGE gel and exposed to autoradiography film for 1 wk. Some variation in expression level from cells with different sites of stable env gene integration is the expected result. The amino acid substitutions in the mutant envelope proteins expressed in the lysates are indicated by their representations in the single-letter amino acid code beneath the headings noting the altered residues. GP+E-86 cells were used as the positive control for expression of wild-type Mo-MuLV envelope protein (w.t.), whereas NIH 3T3 cells were used as the negative control (−). At left are indicated the positions of wild-type uncleaved SU+TM (85 kDa) and SU (70 kDa). There is a cross-reactive protein in the cell lysate that migrates slightly slower than SU+TM (85 kDa).