Figure 4. Codon‐optimization increases the protein concentrations of Mad2 and Mad3.

• A
  Immunoblot of S. pombe protein extracts from cells expressing wild‐type (WT) or codon‐optimized (co) Mad2‐GFP or Mad3‐GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3–5 are a 1:1 dilution series of the extract from cells expressing the codon‐optimized version.
• B
  Immunoblot of protein extracts from wild‐type (WT) or ste13Δ strains probed with antibodies against Mad2, Mad3, and tubulin (loading control). A 1:1 dilution series was loaded for quantification.
• C
  Estimates of the protein concentration relative to wild‐type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Two‐sided t‐tests: P = 0.03 (Mad2‐co), 0.004 (Mad3‐co), 0.82 (Mad2 ste13Δ), 0.15 (Mad3 ste13Δ).
• D
  Whole‐cell GFP concentration from individual live‐cell fluorescence microscopy experiments (a.u., arbitrary units). Boxes show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon‐optimized concentration significantly higher than wild type for both genes (generalized linear mixed model). Mad2‐GFP: n = 468 and 413; Mad2‐co‐GFP: n = 206 and 366; Mad3‐GFP: n = 224 and 127; Mad3‐co‐GFP: n = 160, 450 and 212 cells.
• E
  Representative images from one of the experiments in (D). A single Z‐slice is shown. Cells are outlined in gray.

Source data are available online for this figure.