Journal:

Article Title: Caveolin-1 Is Not Essential for Biosynthetic Apical Membrane Transport

doi: 10.1128/MCB.25.22.10087-10096.2005

Figure Lengend Snippet: Caveolin-2 is retained in the Golgi apparatus in cav1-KD MDCK cells. (A) Control and cav1-KD MDCK cells were grown on Transwell filters for 3 days. Cells were fixed with paraformaldehyde, permeabilized with saponin and immunostained using giantin and caveolin-2 antibodies to visualize the Golgi apparatus and endogenous caveolin-2, respectively. In control cells caveolin-2 was mainly localized at the basolateral membrane. An intracellular perinuclear staining was also seen. In cav1-KD MDCK cells caveolin-2 accumulated in the Golgi, whereas the plasma membrane pool was strongly reduced. (B) Control and cav1-KD cells were grown on filters for 2 days followed by an infection with an adenovirus expressing GFP-cav2 fusion protein. Sixteen hours later, cells were fixed, permeabilized with saponin, and immunostained using antibodies against caveolin-1 and giantin. In control cells GFP-cav2 was found at the basolateral membrane with some additional perinuclear/subapical intracellular staining. In cav1-KD cells, basolateral staining wa s not observed and GFP-cav2 was retained in the Golgi. (C) xz view of GFP-cav2 in filter-grown control and cav1-KD MDCK cells.

Article Snippet: Three to five microliters of OPTIPREP (Axis-Shield) gradient-purified adenovirus in adenovirus storage buffer (5 mM Tris pH 8.0, 50 mM NaCl, 0.05% bovine serum albumin, 60% glycerol) was diluted to 150 μl with OPTIMEM (Gibco BRL) supplemented with l -glutamine.

Techniques: Control, Membrane, Staining, Clinical Proteomics, Infection, Expressing

Journal:

Article Title: Caveolin-1 Is Not Essential for Biosynthetic Apical Membrane Transport

doi: 10.1128/MCB.25.22.10087-10096.2005

Figure Lengend Snippet: Surface delivery of HA and PLAP is unaffected in cav1-KD MDCK cells. (A) Filter-grown (day 3) control and cav1-KD cells were infected with fowl plague virus and pulsed with [35S]methionine. Cells were incubated at 19.5°C to accumulate biosynthetic cargo at the trans-Golgi network, after which the cargo was released and chased for the indicated times at 37°C. The apical surface-exposed HA was quantified as described in Materials and Methods. (B) Filter-grown (days 2 to 3) control and cav1-KD cells were infected with an adenovirus expressing PLAP for 16 h. Cells were pulsed with [35S]methionine and chased for the indicated times. Cell surface proteins were biotinylated and the surface-delivered PLAP was quantified as described in Materials and Methods. The data shown are representative of three (HA) and four (PLAP) independent experiments.

Article Snippet: Three to five microliters of OPTIPREP (Axis-Shield) gradient-purified adenovirus in adenovirus storage buffer (5 mM Tris pH 8.0, 50 mM NaCl, 0.05% bovine serum albumin, 60% glycerol) was diluted to 150 μl with OPTIMEM (Gibco BRL) supplemented with l -glutamine.

Techniques: Control, Infection, Virus, Incubation, Expressing

Journal:

Article Title: Caveolin-1 Is Not Essential for Biosynthetic Apical Membrane Transport

doi: 10.1128/MCB.25.22.10087-10096.2005

Figure Lengend Snippet: GPI-anchored proteins are transported to the cell surface in immortalized mouse embryonic fibroblasts derived from the cav1-KO mice. (A) Coverslip-grown wild-type (wild-type) and cav1-KO MEFs were infected with a PLAP-expressing adenovirus. Ten hours later, cells were fixed and PLAP and caveolin-1 were visualized by immunofluorescence in unpermeabilized (PLAP) or saponin-permeabilized (caveolin-1) cells. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI). (B) Wild-type and cav1-KO MEFs were infected with PLAP-expressing adenovirus for 16 h. Cells were pulsed with [35S]methionine for 15 min and chased for 60 min. Surface-exposed PLAP was biotinylated and quantified as described in Materials and Methods. The data shown come from three independent experiments (C) Wild-type and cav1-KO MEFs were grown on coverslips and fixed, and surface-exposed GPI-anchored proteins (GPI-APs) were labeled using an Alexa 546-conjugated aerolysin mutant (Alexa-546-ASSP) on unpermeabilized cells. Total GPI-anchored protein signal was visualized in paraformaldehyde-fixed, saponin-permeabilized MEFs.

Article Snippet: Three to five microliters of OPTIPREP (Axis-Shield) gradient-purified adenovirus in adenovirus storage buffer (5 mM Tris pH 8.0, 50 mM NaCl, 0.05% bovine serum albumin, 60% glycerol) was diluted to 150 μl with OPTIMEM (Gibco BRL) supplemented with l -glutamine.

Techniques: Derivative Assay, Infection, Expressing, Immunofluorescence, Staining, Labeling, Mutagenesis