Selected article for: "chase 30 min and complete medium"

Title: Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence
  • Document date: 1993_3_1
  • ID: qt44izzh_28
    Snippet: As an additional test for the intracellular localization of proteins having the TGN38 cytoplasmic domain, we examined the production of a soluble form of Tac that is normally shed from the cell surface into the culture medium (Rubin et al., 1985b; Robb and Kutny, 1987; Cullen et al., 1987) . Soluble Tac arises from cleavage of the extracellular domain by a proteolytic activity presumably localized to the plasma membrane (Rubin et al., 1985b; Robb.....
    Document: As an additional test for the intracellular localization of proteins having the TGN38 cytoplasmic domain, we examined the production of a soluble form of Tac that is normally shed from the cell surface into the culture medium (Rubin et al., 1985b; Robb and Kutny, 1987; Cullen et al., 1987) . Soluble Tac arises from cleavage of the extracellular domain by a proteolytic activity presumably localized to the plasma membrane (Rubin et al., 1985b; Robb and Kutny, 1987; Cullen et al., 1987) . COS-1 cells expressing Tac or the Tac-TGN38 chimeric proteins were metabolically labeled and chased for different periods of time. Labeled Tac products were isolated from the cells and from the medium by immunoprecipitation. After 4 h of chase, Tac-expressing cells were found to methionine for 30 min at 37"C and chased for 0, 1, 2, or 4 h in complete medium. Tac proteins were isolated from detergent-solubilized cells and from culture media by immunoprecipitation with a mAb to Tac (7G7). Immune precipitates were analyzed by SDS-PAGE on 10% acrylamide gels. The amount of soluble Tac released into the medium was quantitated and expressed as a percentage of the amount of Tac synthesized during the pulse.

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