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. 2021 Jan 23;22(3):1110. doi: 10.3390/ijms22031110

Figure 5.

Figure 5

Analysis of the activity of different mutant versions of Slt2. (A) Ten-fold serial dilutions of BY4741 slt2Δ-RLM1Myc cells bearing the pRS316 plasmid alone (vector) or expressing Slt2 (WT), Slt2T190A, Slt2Y192F, Slt2T190A Y192A, Slt2ΔC374, Slt2KD, or Slt2CD3. Cells were spotted onto YPD plates in the absence (no stress) or presence of Sodium Dodecyl Sulfate (SDS), tunicamycin (Tuni), Calcofluor White (CFW), caffeine (Caf), zymolyase (Zym), and Congo Red (CR) at the indicated concentrations. (B) The same cells as in (A) were grown to the mid-exponential phase in YPD and then stimulated with 30 µg/mL of CR for 2 h. Cell extracts were resolved by SDS–PAGE, and analyzed by immunoblotting with anti-Slt2-pTpY, anti-Slt2-pT/pTpY, and anti-Slt2-pY/pTpY, as in Figure 1; anti-Mpk1 (total); anti-Myc 4A6; and anti-actin antibodies. (C) Extracts from slt2Δ MKK1-myc (YMJ30 strain) or slt2Δ MKK2-myc (YMJ31 strain) cells transformed with the same plasmids as in (A) were resolved by SDS–PAGE, as in (B), and analyzed by immunoblotting with anti-Myc 4A6 and anti-actin antibodies. (D) β-galactosidase activity of cell extracts from slt2Δ (Y00993) cells co-transformed with pMLP1-LacZ and the same plasmids as in (A) after incubation with 30 µg/mL of CR for 2 h. Data represent the average of the activity of three independent transformants. Error bars indicate the standard deviation. Asterisks (* and **) indicate p-values of <0.05 and <0.01, respectively, assessed by Student’s t-test, and refer to the Slt2 wild type (WT) activity. (E) The same strains as in (C), transformed with the pRS316 plasmid expressing Slt2 (WT) or Slt2CD3. Cells were grown to the mid-exponential phase in YPD and stimulated with 30 µg/mL of CR for 2 h. Cell extracts (input) were incubated with the anti-Mpk1 antibody attached to Dynabeads™ Protein G to purify Slt2 complexes (pull-down) and resolved by SDS–PAGE. Proteins were detected with anti-c-Myc, anti-Mpk1 (Slt2), and anti-actin antibodies by immunoblotting. A control of the Dynabeads-Mpk1 antibody non-incubated with cell extracts is included. A representative assay from three different experiments with distinct transformants is shown.