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. 2004 Feb;24(4):1721–1735. doi: 10.1128/MCB.24.4.1721-1735.2004

FIG. 8.

FIG. 8.

Identification of Med4 and Rgr1 as Kin28 substrates in the PIC. (A) Identification of Med4 as the 39-kDa Kin28 substrate. Substrate candidates were Flag tagged in a wild-type strain background (except Kin28, which directly used Kin28-as-Flag). Nuclear extract made from a Flag-tagged strain (2nd NE) was mixed with Kin28-as nuclear extract (1st NE) in equal amounts. Scaffolds were formed as described in the legend for Fig. 7, loaded onto a 4 to 12% NuPAGE gel, and electroblotted to a polyvinylidene difluoride membrane. Phosphorimaging and Western blotting were performed on the same membrane. The * indicates the position of Med4-Flag on both phosphorimaging and Western blotting. (B) Identification of Rgr1 as the 97-kDa Kin28 substrate. The procedure was the same as for panel A, except samples were separated on a 3 to 8% Tris-acetate gel. The * indicates the position of Rgr1-Flag on both phosphorimaging and Western blotting. In addition, Rpb1 was identified as the 191-kDa Kin28 substrate by H14 antibody in both panels A and B.