FIGURE 6.

HHAT cysteine mapping in selectively permeabilized cells. A, HEK293a cells expressing HHAT-V5-His₆ were permeabilized with 0.04% digitonin (Digi.) or 1% Triton X-100 (TX100) as indicated. Modification was carried out for 30 min at 4 °C with 1 mm mPEG. Samples were analyzed by SDS-PAGE on 7.5% gels, followed by immunoblotting with antibodies to V5 (top panel) or GRP94 (bottom panel). B, HEK293a cells expressing the indicated HHAT single cysteine mutants were permeabilized with 1% Triton X-100. Modification was carried out for 30 min at 4 °C with 1 mm mPEG. Where indicated, DTT was present during the reaction. Samples were analyzed by SDS-PAGE on 7.5% gels, followed by immunoblotting with antibodies to V5 (top panels) or GRP94 (bottom panels). The immunoblots shown are representative results from four independent experiments. C, HEK293a cells expressing HHAT C324A mutant were permeabilized with 1% Triton X-100. Modification was carried out for 30 min at 4 °C with 1 mm mPEG. Where indicated, DTT was present during the reaction. Samples were analyzed by SDS-PAGE on 7.5% gels, followed by immunoblotting with antibodies to V5 (top panel) or GRP94 (bottom panel). HHAT* and GRP94* indicate the positions of the modified proteins.