FIGURE 1. Immunoblot analysis of yeast expressing RTA or E177K either with (A and B) or without (C and D) its signal sequence.
Cells were grown for the hours indicated. 10 μg of fractionated supernatant (S18) and a corresponding volume of fractionated membranes (P18) were separated on SDS-PAGE. Blots were initially probed with polyclonal anti-RTA and subsequently stripped and reprobed with anti-Dpm1p and anti-Pgk1p. Cells expressing RTA (E), E177K (F), mature RTA (G), or mature E177K (H) were grown for the hours indicated in the presence or absence of 1 μg/ml tunicamycin (Tm). Unfractionated total extract (10 μg) was separated on a SDS-polyacrylamide gel. Blots were initially probed with polyclonal anti-RTA and subsequently stripped and reprobed with anti-Dpm1p. Cells harboring the empty vector were analyzed as indicated.