Figure 2.

Cx43 connexons partition into the TX-114 aqueous phase. NRK cells were metabolically labeled with [³⁵S]methionine at 20°C to allow radiolabeled Cx43 to assemble into connexons. The cells were then lysed in 1% TX-114 at 4°C, and the solubilized material analyzed as described below. In A, half of the lysate was subjected to TX-114 phase partitioning at 30°C into detergent-rich (middle panel, D) and detergent-poor aqueous (bottom panel, A) phases before fractionation on 5–20% linear sucrose gradients. The other half of the lysate was fractionated on gradients without prior phase partitioning (top panel, T). Equal fractions were collected from the sucrose gradients and analyzed for Cx43 content by immunoprecipitation followed by SDS-PAGE. In B, one-half of the lysate was TX-114 phase-partitioned into detergent-rich (D) and detergent-poor aqueous (A) phases, and the other half remained unpartitioned (T). The samples were then subjected to chemical cross-linking with DSP (+) or mock cross-linked with DMSO only (−) as indicated, followed by immunoprecipitation of Cx43 and SDS-PAGE. In C, the entire lysate was cross-linked with DSP. One-half of the cross-linked lysate was then TX-114 phase-partitioned into detergent-rich (D) and detergent-poor aqueous (A) phases, whereas the other half remained unpartitioned (T). Cx43 was immunoprecipitated and analyzed by SDS-PAGE. cxon, connexon; m, monomer. Bands migrating at >220 kDa in this and subsequent figures are nonspecifically immunoprecipitated and probably represent binding of newly synthesized fibronectin to the protein A-Sepharose beads (Musil and Goodenough, 1993).