Fig. 3

The vasopressin nonapeptide is necessary for ER aggregation of provasopressin 1–75 and sufficient to aggregate a reporter construct. a Schematic presentation of provasopressin 1–75 (with a C-terminal His₆ tag in black) and its proline/glycine scanning mutants 1–75Pro1–7. b Immunofluorescence localization of example constructs expressed in HN10 cells. The indicated constructs fused to a His₆ tag were transfected into HN10 cells and fluorescently stained using an anti-His₆ antibody. Bar: 10 μm. c Aggregation frequency of these constructs was quantified as before (mean and individual values of four independent transfections, analyzing ~200 expressing cells per transfection). d Transfected cells were lysed, separated by reducing SDS-gel electrophoresis, and subjected to immunoblot analysis to detect intracellularly accumulated provasopressin constructs. Molecular weight standards are indicated in kilodaltons. e Truncated provasopressin sequences 1–75 (positive control), 1–75Pro1 (negative control), 1–50, 1–25, 1–16, and 1–10 were fused to a reporter sequence (Rep) consisting of the C-terminal fragment 101–218 of glutathione S-transferase (GST, orange) with a myc epitope (in purple). f Immunofluorescence localization of indicated constructs expressed in HN10 cells. Bar: 10 μm. g Aggregation frequency of the constructs shown in panel e was quantified as before (mean and individual values of three independent transfections for 1–75-Rep, 1–75Pro1-Rep, and 1–10-Rep, and single determinations for the others, analyzing ~100 expressing cells per transfection)