Figure 2.

The Cx31 mutant 66delD is saponin-insoluble and Triton-soluble, indicating membranous localization. Sequential detergent solubilization of crude lysate of HeLa cells overexpressing (WT)- and (66delD)Cx31-EGFP in saponin followed by Triton X-100 then SDS revealed no Cx31 protein is saponin-soluble which would be indicative of a cytosolic pool (in contrast to the unfused EGFP protein). The presence of (66delD)Cx31-EGFP protein solely in the Triton-soluble fraction indicates that the protein is mainly present on membranous structures. The additional presence of a Triton-insoluble pool of (WT)Cx31-EGFP (present in the SDS-soluble fraction) is indicative of gap junction aggregates. The LAMP1 and GAPDH control proteins are present in the Triton- and saponin-soluble fractions, respectively, as expected.