Figure 3.

Pmt1 and Pmt2 protein levels are reduced in BFR1 deficient cells. Western blot analysis of protein levels of Pmt1 (A), Pmt2 (B), and other representative proteins known to be targeted by Bfr1 [42] (C) in total cell extracts from wild type (BY4741), JEY06 (wild type ER-GFP), bfr1Δ and bfr1Δ ER-GFP strains. (A) and (B) left panels: 20 μg of protein were resolved on a 12% PAA gel and detection was performed with an anti-Pmt1 and anti-Pmt2 antibody respectively. Pmt2 detection was initially performed with a polyclonal serum detecting Pmt1 at the same time (lower and upper band indicated by black arrows respectively). In subsequent experiments preabsorption of the polyclonal serum was performed on membranes from PMT2 deficient cells (single band detection for Pmt2 in e.g., Figure 4). G6PDH was used as loading control. (A) and (B) right panels: Western blot signals were quantified using Image Studio Lite v 5.2 and PMT signals were first normalized to the respective G6PDH signals and subsequently normalized to the Pmt/G6PDH ratio calculated for wild type cells. Error bars represent the range of values from three independent experiments. For statistical significance one-sample t-test was performed on log₂(fold change). (C) 20 μg of protein were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. G6PDH was used as loading control and results are representative of three independent experiments. Asterisks report on statistical significance: * p ≤ 0.05, ** p ≤ 0.01.