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. 2019 Dec 10;20(24):6220. doi: 10.3390/ijms20246220

Figure 4.

Figure 4

Pmt2 overexpression partially rescues loss of ER-GFP O-mannosylation in bfr1Δ cells. (A) Western blot analysis of ER-GFP O-mannosylation in total cell extracts from JEY06 (wild type ER-GFP) and bfr1Δ ER-GFP strains transformed with either pRS41N (empty vector) or pJC09 (PMT2). Strains were grown under standard conditions in YPD supplemented with nourseothricin for selection. Twenty micrograms of protein were resolved on a 12% PAA gel and detection was performed with an anti-GFP antibody. G6PDH was used as loading control. Arrows on the right indicate the main GFP signal (black arrow) and signals emanating from higher O-mannosylated GFP fractions (white arrow). Pmt2 overexpression partially restores ER-GFP O-mannosylation in the bfr1Δ strain. (B) Flow cytometry analysis of strains described in (A). Fluorescent signal for each strain resulted from analysis of 20,000 cells and statistical significance was assessed by a 2way ANOVA on three independent experiments. Pmt2 overexpression partially restores ER-GFP fluorescence to the level detected in the JEY06 strain. (C) Western blot analysis of Pmt2 protein levels in total cell extracts from wild type (BY4741), bfr1Δ, JEY06 (wild type ER-GFP), and bfr1Δ ER-GFP strains transformed with either pRS41N (empty vector) or pJC09 (PMT2) and grown as in (A). Twenty micrograms of protein were resolved on a 12% PAA gel and detection was performed with an anti-Pmt2 antibody. G6PDH was used as loading control. N.s. = not significant. Asterisks report on statistical significance: * p ≤ 0.05.