FIG. 2.

Analysis of membrane-associated M1 expressed alone by RVV and in influenza virus-infected cells after TX-100 detergent treatment. (A) HeLa cells (5 × 10⁶) were infected at an MOI of 10 with RVVM1. At 6.0 hpi, infected cells were labeled for 30 min with 400 μCi of ³⁵S Easy Tag and chased for 90 min. Infected cells were harvested and fractionated for 4K supernatant. The membrane fractions were isolated from the 4K supernatant with a floatation gradient as described in Materials and Methods and were either mock treated or treated with varying concentrations (0.01, 0.02, 0.03, 0.04, 0.05, and 0.06%) of TX-100 detergent for 15 min on ice. Each sample was then analyzed again by floatation in sucrose gradients. Gradient fractions were immunoprecipitated using rabbit anti-WSN polyclonal antibodies and analyzed by SDS–10% PAGE. (B and C) Influenza virus-infected cells were labeled at 6.5 hpi for 30 min and chased for 90 min. The total 4K supernatants were prepared and analyzed with floatation gradients before (−) and after (+) TX-100 treatment at different concentrations. Note that both HA and M1 decreased in the membrane fractions (fractions 1 and 2) after treatment with higher TX-100 concentrations (0.06 and 0.08%). Results in panels B and C are from two separate experiments.