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Figure 9


Fig. 9. Co-immunoprecipitation studies on the effect of TNF{alpha}+CHX on PKC{zeta}-MHCIIB interaction. (A) TtT/GF cell lysates were prepared from untreated cells (L). Precleared cell lysates were incubated with proteinA-Sepharose4B beads that had been preincubated with either buffer alone (B) or anti-vimentin (Vm) or anti-MHCIIB (MHCIIB). The pellets were subjected to SDS-PAGE followed by immunoblotting with anti-PKC{zeta} antibody. A representative membrane shows the presence of a 70 kDa immunoreactive band in the cell lysate and in the MHCIIB immunoprecipitate. (B) Untreated TtT/GF cell lysates (L) were prepared. Precleared cell lysates were incubated with proteinA-Sepharose4B beads that had been preincubated with either buffer alone (B), anti-vimentin (Vm) or anti-PKC{zeta} (PKC{zeta}) antibodies. The pellets were subjected to SDS-PAGE followed by immunoblotting with anti-MHCIIB. A representative membrane shows the presence of a 210 kDa immunoreactive band in the cell lysate and in the PKC{zeta} immunoprecipitate. (C) Control and TtT/GF cells treated for 24 hours with TNF{alpha}+CHX were subjected to immunoprecipitation with PKC{zeta} antibody. The pellets were recovered and subjected to western blotting with MHCIIB antibodies. This representative membrane shows presence of a 70 kDa immunoreactive band in the immunoprecipitates of both untreated and treated cells.