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. 2015 Oct 9;5:14909. doi: 10.1038/srep14909

Figure 4. The Rpt6 tail exhibits a distinct functional relationship with Nas6 in vivo.

Figure 4

(a) Phenotypic analysis showing effect of nas6Δ on the growth of rpt6-Δ1 or rpt3-Δ1 single, or double mutants. Four-fold serial dilutions of indicated cells were spotted onto YPD plates, synthetic complete medium (SC), or SC medium containing canavanine (1 μg/ml), and incubated for 2–3 days at the indicated temperature. For testing sensitivity to canavanine (an arginine analog), arginine was omitted from the SC medium. (b) Effect of nas6Δ on the degradation of polyubiquitinated proteins in rpt6-Δ1 or rpt3-Δ1 single, or double mutant cells. The cells were cultured for 6 hours at 37 °C. Whole cell lysates (20 μg) were subjected to 10% Bis-Tris SDS-PAGE for immunoblotting (IB) of polyubiquitinated proteins, and 12% SDS-PAGE for immunoblotting of Nas6 and proteasome subunits. Rpt3 and Rpt6 are base subunits. Rpn8 is a lid subunit. Pgk1 serves as a loading control. Lighter exposure of anti-ubiquitin (Ub) immunoblot is shown at right to further illustrate the difference in polyubiquitinated protein levels. Molecular weight markers are at left in kDa.