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. 1997 Dec 29;139(7):1735–1745. doi: 10.1083/jcb.139.7.1735

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Aggregation of furin-FLAG in NH₄Cl-treated cells. Stably transfected RBL cells expressing furin-FLAG, Tac, or Tac-DKQTLL were pulse labeled for 30 min with [³⁵S]methionine and chased for 0 h (A, D, and G), 4 h (B), or 6 h (C, E, F, H, and I) in the absence (A, B, D, E, G, and H) or presence (C, F, and I) of 50 mM NH₄Cl. Detergent lysates of the cells were fractionated by sedimentation on 5–20% sucrose gradients for 16 h (see Materials and Methods). Individual gradient fractions (1– 15) were immunoprecipitated with either an antifurin antibody (Fur2) (A–C) or an antibody to Tac (7G7) (D–I), and then analyzed by SDS-PAGE and fluorography. The MHC class I H-2K^b/β₂-microglobulin complex (∼57 kD, fraction 3) and the transferrin receptor (∼190 kD, fractions 5 and 6) were used as internal size standards for integral membrane proteins. Gradient fraction numbers, from top to bottom, are indicated. The bracket in C indicates high molecular weight aggregates of furin-FLAG.

Aggregation of furin-FLAG in NH₄Cl-treated cells. Stably transfected RBL cells expressing furin-FLAG, Tac, or Tac-DKQTLL were pulse labeled for 30 min with [³⁵S]methionine and chased for 0 h (A, D, and G), 4 h (B), or 6 h (C, E, F, H, and I) in the absence (A, B, D, E, G, and H) or presence (C, F, and I) of 50 mM NH₄Cl. Detergent lysates of the cells were fractionated by sedimentation on 5–20% sucrose gradients for 16 h (see Materials and Methods). Individual gradient fractions (1– 15) were immunoprecipitated with either an antifurin antibody (Fur2) (A–C) or an antibody to Tac (7G7) (D–I), and then analyzed by SDS-PAGE and fluorography. The MHC class I H-2K^b/β₂-microglobulin complex (∼57 kD, fraction 3) and the transferrin receptor (∼190 kD, fractions 5 and 6) were used as internal size standards for integral membrane proteins. Gradient fraction numbers, from top to bottom, are indicated. The bracket in C indicates high molecular weight aggregates of furin-FLAG.