![]() The lower panel depicts whole-cell lysates which were subjected to IB analysis using antibodies to caveolin as control for the purity of the S100 fractions. C and M fractions were prepared and subjected to IP followed by IB with antibodies to Flag. 3T3-JAMP and 3T3-N1 cells were either untreated (lanes 1 to 4)or treated with MG132 (lanes 5 to 8) or TM (lanes 9 to 12). (e) JAMP is actively degraded in the cytoplasm. The lower panel depicts the same membrane stripped and reprobed with anti-caveolin antibody as a control to verify the purity of the membrane fraction. Lanes 3 and 4 represent whole-cell lysates of the indicated cell types. Proteins (1 mg) were subjected to IP (lanes 1 and 2) followed by IB with anti-Flag antibody. 3T3-JAMP cells were grown and proteins were prepared as cytosolic (C) and membrane (M) fractions (see Materials and Methods). (d) JAMP localization within the S100 fraction. These mutants were transfected into 293T cells, and their expression patterns were monitored by immunoblot analysis. Two putative N-glycosylation sites were mutated as JAMP N1 (aa 22)- or JAMP N2 (aa 134)-GFP, respectively. (c) JAMP is N glycosylated on an NH 2 site. Immunoprecipitates were subjected to Endo-H treatment (5 min) followed by immunoblot analysis with anti-Flag antibody. 3T3-JAMP cells were fractionated into cytosolic and membrane proteins which were than used for immunoprecipitation of JAMP using antibodies to Flag. (b) Deglycosylation enzyme alters the pattern of JAMP expression. Proteins were immunoprecipitated (IP) and immunoblotted (IB) with anti-Flag antibody. 3T3-JAMP or 3T3 control cells were treated for 16 h with TM (1 μg/ml) or thapsigargin (0.25 μM). (a) JAMP exhibits two major forms which are altered in response to stress. JAMP is N glycosylated and is primarily localized within the insoluble S100 membrane fraction. ![]()
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