Fig. 13

SDS-PAGE analysis of trypsin-, salt-and NEM-sensitive proteins in adipocytes.

Isolated rat adipocytes, which had been metabolically labeled with [³⁵S]methionine (lanes 1–3) or left unlabeled (lanes 4–6), were incubated (20 min, 22^°C) with 100 µg/ml trypsin (lanes 1, 2, 4, 5) or left untreated (lanes 3, 6). After termination of the digestion, the cells were adjusted to 0.5 M NaCl (lanes 2, 3, 5, 6) or left untreated (lanes 1, 4) and then separated from the incubation medium by flotation. The infranatant below the cell layer was removed and precipitated with PEG. Equivalent portions (with regard to the number of adipocytes used for the pre-treatment) of the [³⁵S]methionine-labeled precipitates were directly dissolved in sample buffer (lanes 1–3), whereas those of the unlabeled precipitates were treated with [¹⁴C]NEM (lanes 4–6) as described in Materials and Methods. All samples were analyzed by SDS-PAGE and fluorography. The flu-orogram shows the results of two independent extract preparations and labelings with separate adipocyte preparations each. The molecular weights indicated were derived from marker proteins run in parallel on the same gel.