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Fig. 1. Expression level and membrane association of HASPB-GFP fusion proteins expressed in CHO cells. (A) CHO cells expressing HASPB-GFP fusion proteins as indicated were grown on six-well plates in the absence (lanes 1, 3, 5 and 7) or presence (lanes 2, 4, 6 and 8) of 1 µg/ml doxicycline (dox). Cells were detached and collected by centrifugation followed by lysis in SDS sample buffer. 1% of each lysate corresponding to cells from one well were subjected to SDS-PAGE. Following SDS-PAGE and western blotting HASPB-GFP fusion proteins were detected using affinity-purified anti-GFP antibodies. (B) CHO cells expressing HASPB-GFP fusion proteins as indicated were fractionated into cytosolic (lane 1) and membrane fractions (lane 2). Additionally, the membrane fraction was subjected to carbonate extraction resulting in a carbonate supernatant containing loosely bound proteins (lane 3) and a carbonate pellet containing proteins tightly associated with membranes (lane 4). 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.
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