Journal: Molecular & Cellular Proteomics : MCP
Article Title: Stable Isotope Labeling by Amino Acids in Cell Culture and Differential Plasma Membrane Proteome Quantitation Identify New Substrates for the MARCH9 Transmembrane E3 Ligase *
Figure Lengend Snippet: Mechanism of substrate down-regulation. A , FcγRIIB or SLAM were co-transfected together with GFP-tagged wild-type or tryptophan to alanine mutant ( W/A ) MARCH9 into 293T cells and stained with antibodies specific for FcγRIIB or SLAM. The percentage of cells in each quadrant is shown. B , pulse-chase analysis of metabolically labeled HA-tagged SLAM in the presence or absence of MARCH9. HA-tagged SLAM-expressing 293T cells in the presence or absence of MARCH9 were [ 35 S]methionine-radiolabeled for 20 min and chased for the times indicated. Triton X-100 lysates were immunoprecipitated with HA-specific monoclonal antibody and treated with Endo H. Samples were analyzed by SDS-PAGE and autoradiography. The half-life ( t ½) of the Endo H-sensitive, immature form and the Endo H-resistant, mature form of SLAM is shown. C , Tsg101 siRNA-depleted 293T cells were co-transfected with SLAM and GFP-tagged MARCH9. SLAM and MARCH9 expression were analyzed by flow cytometry and compared with mock-depleted cells or cells transfected with tryptophan to alanine mutant MARCH9 ( W/A ). IP , immunoprecipitation.
Article Snippet: The cells were lysed in 1% Triton X-100 and precleared twice with CL-4B Sepharose beads (Sigma-Aldrich), and HA-tagged SLAM was precipitated with the HA.11 monoclonal antibody (clone 16B12, Covance) and protein A-Sepharose for 1 h, washed three times with 0.5% Triton X-100 in PBS, eluted in sample buffer, and heated to 95 °C for 10 min. For endoglycosidase H (Endo H) digestion samples were supplemented with 250 units of Endo H (New England Biolabs) and incubated at 37 °C for 1 h.
Techniques: Transfection, Mutagenesis, Staining, Pulse Chase, Metabolic Labelling, Labeling, Expressing, Immunoprecipitation, SDS Page, Autoradiography, Flow Cytometry, Cytometry