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. 2014 Apr 11;6(4):1349–1361. doi: 10.3390/toxins6041349

Figure 2.

Figure 2

Analysis of PAP expression and ribosome depurination in yeast cells co-transformed with PAP and L3Δ99. (A) Immunoblot analysis of PAP expression. Cells were induced for 6 h with galactose. Total cell extracts (15 μg) were electrophoresed through 12% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and probed with PAP polyclonal antibody (1:2000). The membrane was subsequently stripped and re-probed with G6PD monoclonal antibody (1:5000) as a loading control; (B) Analysis of ribosome depurination in yeast expressing PAP, PAPE167V and PAP/L3Δ99 by primer extension analysis. The depurination primer (Dep) was used to measure the extent of depurination and the 25S primer (25S) was used to measure the amount of 25S rRNA; (C) In vitro depurination analysis of yeast rRNA using PAP protein isolated from yeast cells co-transformed with PAP/L3Δ99, compared to purified PAP protein (PAP). CK is untreated rRNA.