Figure 2
From: C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex
Processed forms of Spt7 in yeast deletion strains. A. Wild-type (BY4741) or the indicated yeast deletion strains (gcn5Δ0, rtg2Δ0, spt8Δ0, ubp8Δ0, ada1Δ0, spt20Δ0; lanes 3–8, respectively) were transformed with YCpDed-TAP-Flag-SPT7, with the exception of lane 1 (ctrl) which was transformed with the control vector YCp-TAP-Flag. Cells were grown to an A600 = 1.0–1.5 in YP containing 2% glucose and crude cell lysates prepared by glass bead disruption. 50 μg of protein was separated by 5% SDS-PAGE and Western blotted with anti-Flag antibody. The mobility of a 200 kDa molecular weight marker is indicated on the left. Spt7SAGA and Spt7SLIK are marked. B. BY4741 (Wt, lane 2) and the indicated deletion strains (ubp8Δ0, lane 3; sgf11Δ0, lane 4; sgf29Δ0, lane 5; sgf73Δ0, lane 6) containing TAP-Flag-Spt7 were grown as above, crude yeast extracts separated by SDS-PAGE and Western blotted with anti-Flag-antibody. Lane one contains crude extract from the wild-type strain BY4741. Spt7SAGA, Spt7SLIK and Spt7Form3 are marked. C. Wild-type (BY4741 lanes 2 and 3) or the indicated yeast deletion strains, gcn5Δ0 (lane 4), spt8Δ0 (lane 5), spt20Δ0 (lane 6), ada1Δ0 (lane 7), rtg2Δ0 (lane 8), ubp8Δ0 (lane 9) containing YCpDed-TAP-Flag-SPT7 were grown in YP broth containing 2% glucose. Lane 1 (ctrl) contains BY4741 transformed with the control vector YCp-TAP-Flag. TAP-Flag-Spt7 was purified through affinity chromatography on IgG-Agarose and eluted after cleavage with TEV protease. Approximately equal amounts of Spt7 were separated by 5% SDS-PAGE and Western blotted with anti-Flag antibody. The mobility of 250 and 150 kDa molecular mass markers are indicated on the left. Lane 3 contains one half of the wild-type sample applied in lane 2 to facilitate quantitation.