FIG. 2.

sHyal2, but not low pH, promotes shedding of JSRV SU. (A and B) 293 cells expressing JSRV Env were metabolically labeled for 1 h and then chased for 3 h before the addition of the indicated amounts of sHyal2. Cells were chased for an additional 3 h before being lysed. Cell lysates and culture media were harvested and immunoprecipitated using anti-FLAG beads. Samples were resolved by SDS-PAGE and analyzed by autoradiography. (A) Env expression and processing in cell lysates. (B) JSRV SU shedding into the culture medium. (C and D) Metabolic labeling was performed as described for panels A and B, except that cells were incubated in the absence or presence of 5 μg sHyal2. The culture medium was harvested (pre-pH), and the cells were treated with the indicated pH buffers for 5 min at 37°C. The pH buffers were harvested and neutralized (during pH), and the cells were cultured for an additional 1 h. The culture medium was harvested (post-pH), and the cells were lysed. The cell lysates (C) and harvested supernatants or neutralized pH buffers (D) were immunoprecipitated using anti-FLAG beads and were analyzed by autoradiography. Env, JSRV Env precursor; NC, parental 293 cells not expressing JSRV Env. The strong band appearing in the parental 293 cells (panels A and C, lanes 1), and sometimes in the Env-expressing 293 cells, is likely a cellular protein that was nonspecifically pulled down by anti-FLAG beads.