Article Figures & Data
Figures
- FIG. 1.
Expression of EYFP-p48 and EYFP-p48ΔTM in COS-7 cells. (A) Cells were transfected with pEYFP-p48, pEYFP-p48ΔTM, or pEYFP-TM and observed 24 h posttransfection. Magnification is ×400. (B) Cell lysates prepared from COS-7 cells transfected with the indicated plasmid were separated by SDS-10% PAGE and subjected to Western immunoblotting with anti-GFP-polyclonal antibody.
- FIG. 2.
In vitro-translated p48 binds GST-VAP-A in a pull-down assay. 35S-p48 was incubated with glutathione-Sepharose 4B beads only, bead-immobilized GST, or GST-VAP-A. The bound radiolabeled protein was eluted and analyzed by SDS-10% PAGE and autoradiography. The amount of 35S-p48 in the Input lane represents the total amount of protein added to each pull-down reaction.
- FIG. 3.
EYFP-p48 and EYFP-p48ΔTM coimmunoprecipitate with Myc-tagged VAP-A. (A) EYFP-p48 or EYFP-p48ΔTM and Myc-tagged VAP-A were coexpressed in COS-7 cells. Twenty-four hours posttransfection, the cell lysates were subjected to immunoprecipitation in the presence or the absence of anti-c-Myc monoclonal antibody. The immunoprecipitates were separated by SDS-PAGE and then subjected to immunoblotting with rabbit anti-GFP antibody. (B) pEYFP-p48 or pEYFP-p48ΔTM and pcVAP-A were transfected in separate cultures of COS-7 cells. The cell lysates were mixed and then subjected to immunoprecipitation as described above.
- FIG. 4.
p48 behaves as an integral membrane protein in COS-7 cells. Lysates of COS-7 cells transfected with EYFP-p48, EYFP-p48ΔTM, or VAP-A were treated with sodium carbonate, Triton X-100, or Triton X-114, as described in Materials and Methods. Proteins in the Control lane received no treatment. Pellets (P) and supernatants (S) from each sample were separated by SDS-PAGE, followed by Western blotting with the appropriate antibody.
- FIG. 5.
p48 and VAP-A cofractionate with ECFP-RhoB. COS-7 cells transfected with pcVAP-A and pEYFP-p48 (A) or pcVAP-A and pECFP-RhoB (B) were subjected to subcellular fractionation on sucrose gradient as described in Materials and Methods. Sucrose gradient fractions were analyzed by Western immunoblotting with c-Myc monoclonal antibody for cVAP-A and ECFP-RhoB and GFP polyclonal antibody for EYFP-p48. Sedimentation is from left to right (lanes 1 to 8). The lane labeled P is the membrane pellet not subjected to fraction procedures.
- FIG. 6.
Expression of EYFP-p48 prevents cell surface expression of VSV G protein. COS-7 cells were transfected with pcVSV-G and pEYFP (A), pcVSV-G and pEYFP-48 (B), or pcVSV-G and pEYFP-p48ΔTM (C). VSV G was detected 24 h posttransfection by indirect immunofluorescence with an anti-c-Myc monoclonal antibody, followed by tetramethyl rhodamine isocyanate-conjugated secondary antibody. Magnification is ×400.
- FIG. 7.
VSV G protein acquires endoglycosidase H resistance in the presence of EYFP-p48. COS-7 cells were cotransfected with pEYFP or pEYFP-p48 and pcVSV-G. The cells were labeled for 2 h with TRANS 35S-label followed by a 2-h chase period. VSV G protein was immunoprecipitated from cell lysates with c-Myc monoclonal antibody and treated with either endoglycosidase H (A) or PNGase F (B).
