FIG. 4.

Formation of PCC with mutant Psc proteins. (A) Approximately 800 ng PCC containing mutant Psc proteins was separated on a 4 to 15%-gradient SDS-PAGE gel and stained with colloidal Coomassie. The identity of the Psc subunit of the complex is indicated [e.g., PCC (e23) for PCC containing Psc^e23 protein]. Positions of mutant Psc (circles) and other subunits are indicated. *, degradation product of Ph; **, Hsp70. (B) Western blots to assess relative stoichiometry of PCC subunits in complexes formed with mutant Psc (1 pmol in blots with anti-Pc or anti-dRing1; 100 fmol in blots with anti-Psc or anti-FLAG). PCC containing the Psc^e24 or Psc¹ protein was probed with the 6E8 antibody, while all others were probed with the 7E10 antibody. Because equal amounts of protein were loaded in each lane, intensities of bands for each antibody should be similar in all lanes if PCC subunit stoichiometries are similar in all complexes. The lower region of the gel probed with 7E10 is not shown because it was probed with another antibody. (C) PCC (1 μg) carrying mutant Psc proteins was separated on a 4 to 15%-gradient SDS-PAGE gel and stained with colloidal Coomassie. (D) Two hundred nanograms PCC carrying mutant Psc proteins was separated on 8% SDS-PAGE gels, and the Western blots were probed with antibodies against PCC subunits or with an anti-FLAG antibody to detect Psc. (E) Insect cells were infected with viruses encoding FLAG-tagged PSC^456-1603 and other PCC subunits, and immunoaffinity purification was performed on nuclear extracts from infected cells. Input (I), flow-through (FT), and elution (E) samples were analyzed for the presence of PCC subunits by Western blotting.