Figure 3. Expression of sams‐3, sams‐4, and sams‐5 is negatively regulated by SAM synthetase activity through alternative splicing.

• A
  Alternative splicing of sams‐3, sams‐4, and sams‐5 in smg‐2 (yb979) and smg‐2 (yb979); sams‐5 (gk147); sams‐1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S‐complete medium alone (lanes 1 and 5), with OP50 (OD₆₀₀ = 10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L‐Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 h at 20°C. The splicing patterns were analyzed and presented as in Fig 2B (n = 3).
• B, C
  Western blot analysis of SAMS‐1, SAMS‐3, and SAMS‐4 during larval development in the smg‐2 (yb979) (B) and wild‐type (C) backgrounds. Genotypes of the worms are smg‐2 (w) and smg‐2; sams‐5; sams‐1 (s) in (B) and wild‐type (w) and sams‐1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti‐β‐tubulin (B) or Coomassie Brilliant Blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix␣Fig S12. Note that upregulation of SAMS‐1 protein in the wild type during larval development in (C) is consistent with feeding‐induced upregulation of sams‐1 mRNA in Fig EV3D.