Table 1.
Primers used for cloning and mutagenesis.
Fig 1.
Murine and rat IGSF1-2 are glycoproteins.
A) CHO cells were transfected with empty vector (pcDNA4), wild-type murine IGSF1-2-Myc/His, or murine IGSF1-2 (N43Q)-Myc/His expression vectors. Whole cell protein lysates were collected and treated with PNGaseF (P), EndoH (E), or buffer alone (-) before being subjected to SDS-PAGE and immunoblotting (IB) with a Myc antibody. Molecular weight markers (in kDa) are shown at the left. Lanes are numbered at the bottom. B) CHO cells were transfected and lysates analyzed as in panel A, with the following exception: wild-type rat IGSF1-2-Myc/His was used in place of the mutant murine expression vector. *, non-specific band.
Fig 2.
Murine and rat IGSF1-2 proteins are not secreted from transfected cells.
HEK293 cells were transfected with expression plasmids for wild-type (WT) or glycosylation mutant (NQ) forms of murine or rat IGSF1-2, murine transthyretin (TTR), or empty vector (pcDNA4). Note, in all cases, proteins were expressed with Myc/His tags at their C-termini. Media (top two panels) and whole cell protein lysates (bottom panel) were collected and analyzed by SDS-PAGE and immunoblotting for Myc. In the middle panel, proteins in culture medium were analyzed directly. In the top panel, proteins in the media were analyzed following Ni-NTA purification and enrichment.
Fig 3.
Murine and rat IGSF1-2 proteins do not traffic to the plasma membrane of transfected cells.
A) HEK293 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine BMP type IA receptor (BMPR1A), or empty vector (pcDNA4). Note, IGSF1-2 proteins were expressed with Myc/His tags at their C-termini, whereas BMPR1A had the Myc tag alone. Cell surface proteins were labeled with biotin prior to collection of whole cell lysates. Lysates were immunoprecipitated (IP) with Myc-beads and then subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total proteins were immunoblotted with anti-Myc (bottom), whereas biotinylated proteins were identified with streptavidin-HRP (top). B) HEK293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His. Cells were then fixed and subjected to immunofluorescence with a Myc antibody (green) under non-permeabilizing (top) and permeabilizing conditions (bottom). C) Cells were transfected as in panel B, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP-78/BiP (red). The overlay is shown in yellow. In B and C, nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.
Fig 4.
IGSF1-2 is not secreted from homologous HepG2 cells.
A) Expression of endogenous IGSF1-CTD in HepG2 cells as determined by immunoblot using an antibody that recognizes the N-terminus of the CTD (black arrow). Protein lysates were treated with PNGaseF to confirm the specificity of the signal (gray arrow). B) HepG2 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine transthyretin (TTR), or empty vector (pcDNA4). In all cases, proteins were expressed with Myc/His tags at their C-termini. Media was collected and His-tagged proteins purified by Ni-NTA chromatography before analysis by SDS-PAGE and immunoblotting for Myc (top). Cell protein lysates were collected and immunoprecipitated (IP) with Myc-beads before analysis by Myc immunoblot (bottom). Note that the non-specific band (*) partially obscures the rat IGSF1-2 in the bottom panel (third lane).