Fig. 7. Expression level, membrane association and post-translational acylation of HASPB-N18-GFP in CHO wild-type cells and CHO mutant K3 cells. (A) CHO wild-type cells and CHO K3 cells (both expressing HASPB-N18-GFP) were grown on six-well plates to about 80% confluency in the absence (lane 1) or presence (lane 2) of doxicycline (1 µg/ml) for 48 hours at 37°C. Cells were detached with PBS/EDTA, collected by centrifugation and lysed in SDS sample buffer. 1% of each lysate corresponding to cells from one well were subjected to SDS-PAGE. HASPB-N18-GFP was detected by western blotting using affinity-purified anti-GFP antibodies. (B) CHO wild-type cells and CHO K3 cells (both expressing HASPB-N18-GFP) were grown on six-well plates to about 80% confluency in the presence of 1 µg/ml doxicycline for 48 hours at 37°C. Subcellular fractionation and carbonate extraction of membranes was performed and 5% of each fraction was combined with SDS sample buffer and proteins were separated by SDS-PAGE. Following western blotting, HASPB-GFP fusion proteins were detected with affinity-purified anti-GFP antibodies. (C) CHO wild-type cells, CHO K3 cells (both expressing HASPB-N18-GFP) as well as control cell lines expressing HASPB-N18-GFP myr/palm and HASPB-N18-GFP palm, respectively, were grown on six-well plates to about 80% confluency in the presence of 1 µg/ml doxicycline for 48 hours at 37°C and labelled with [³H]myristate and [³H]palmitate. Cell lysates were prepared and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Immunopurified fractions were split into two samples, separated on SDS gels and either processed by fluorography (upper panel) or silver staining (lower panel).