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Fig. 8. NCAM is mono-ubiquitylated at the plasma membrane. (A,B) After either no induction (–) or induction of NCAM endocytosis for 60 minutes (+) in B35, NCAM140-expressing (A) or NCAM180-expressing (B) B35 cells, lysates were subjected to immunoprecipitation with an NCAM-specific antibody. Immunoblot analysis was carried out using an antibody specifically recognizing ubiquitin (P4D1, top). After antibody removal the blot was reprobed with an NCAM-specific antibody (123C3) as a control (bottom). (C) Endocytosis of NCAM140 was induced (+) for 60 minutes or not induced (–) in NCAM140-expressing B35 cells. Cell lysates were immunoprecipitated with an NCAM-specific antibody. Immunoblot analysis was carried out using an antibody recognizing specifically poly-ubiquitylated proteins (FK1). After antibody removal the blot was reprobed with an antibody recognizing mono- and polyubiquitylated proteins (FK2). After another antibody removal an incubation with an NCAM-specific antibody (123C3) was carried out as control. To control the functionality of the antibodies whole-cell lysates (L) were applied to the gel. (D) After no induction (–) or induction of NCAM endocytosis for 60 minutes (+) in NCAM140-expressing B35 cells, cell surface proteins were biotinylated at 4°C (lanes 1 and 2). After stopping the reaction, lysates were immunoprecipitated with an NCAM-specific antibody. Immunoprecipitated NCAM was eluted from the sepharose beads and a second precipitation was carried out using streptavidin-agarose beads to isolate cell surface NCAM proteins. In lanes 3 and 4, control immunoprecipitations were are shown with total NCAM [lanes 1 and 3 without (–) and lane 2 and 4 with (+) induction of endocytosis]. Immunoblot analysis was performed using an antibody specifically recognizing ubiquitin (P4D1, top). After antibody removal the blot was reprobed with NCAM-specific antibodies (123C3) as a control (bottom).
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