Figure 9.

CMTX-linked Cx32 mutants do not assemble connexons. PC12 cells expressing WT Cx32 (lanes 3 and 4), mutant Cx32 (lanes 5–10), or no Cx32 (lanes 11 and 12) were metabolically labeled for 20 min at 37°C and then 4.6 h at 20°C as described in MATERIALS AND METHODS. Alternatively, MH₁C₁ cells (lanes 1 and 2) were labeled for 4 h at 37°C. Triton X-100-solubilized cell extracts were cross-linked with 1 mM EGS for 30 min at 4°C (lanes 2, 4, 6, 8, 10, and 12), or treated with DMSO only for a mock–cross-linked control (lanes 1, 3, 5, 7, 9, and 11). Cx32 was immunoprecipitated and analyzed by SDS-PAGE on 7.5–12.5% gradient gels. The positions of monomeric Cx32 (m) and cross-linked Cx32 connexons (cxon) are indicated at the left. The bands migrating slower than monomeric Cx32 in non–cross-linked mutant immunoprecipitates are mainly SDS-induced aggregates of Cx32.