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Figure 4


Fig. 4. Polyubiquitylated SCD1 accumulates in the ER in the presence of MG132. (A) CHO-K1 cells were transfected with pcDNA3.1 or the plasmid harboring SCD1-HA cDNA, and grown for 48 hours. DMSO or 50 µM MG132 was then added to the cells and the culture continued for 5 hours. Post-nuclear supernatant (PNS) was prepared from the cells, and then fractionated into the supernatant (S) and membrane (P) fractions. Both fractions were subjected to immunoprecipitation using anti-HA antibody and the immunoprecipitates were then analyzed by SDS-PAGE and subsequent immunoblotting using anti-polyubiquitin antibody (upper panel). The filter was re-blotted with anti-HA antibody (middle panel), or anti-p97 antibody (lower panel). Lanes 1-4: 5% of PNS prepared from the cells grown under the indicated conditions were directly applied. Lane 13 (IP control): immunoprecipitation without cell lysate. (B) Quantification of p97 recovered to the ER of the SCD1-HA-expressing cells. Immunoprecipitation was performed as described in A, the gel was analyzed by digital autoradiography, and band intensities were calculated setting that of the total amount of p97 as 100%. The average of three independent experiments is shown.





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