Figure EV1. MAP3Ks activated by osmotic shock.

• A
  Schematic of the haploid genetic screen workflow to map genetic regulators of p38 activation in response to osmotic shock.
• B
  HAP1 cells and cells deleted for ZAK (ΔZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies.
• C
  U2OS cells stably expressing S‐HA‐ZAKβ were treated with increasing concentrations of sorbitol (1 h). Lysates were analyzed as in (B).
• D
  U2OS cells were pretreated with ZAK inhibitor (ZAKi, 0.5 h) and treated with sorbitol (500 mM, 1 h). Lysates were treated with lambda phosphatase (λ phos.) and phosphatase inhibitor (PPi) as indicated and analyzed as in (B).
• E
  U2OS ΔZAK cells stably expressing wildtype (WT) or kinase‐dead (KD) ZAKβ‐GFP were treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed as in (B).
• F
  U2OS and ΔZAK cells were treated with sorbitol (500 mM) for the indicated time points and analyzed as in (B).
• G
  U2OS and ΔZAK cells were treated with sorbitol (500 mM), washed out for sorbitol, and allowed to recover for the indicated time points. Lysates were analyzed as in (B).
• H
  U2OS and ΔZAK cells were transfected with siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed as in (B).
• I
  U2OS, ΔZAK, and ΔZAK cells rescued with wildtype (WT) or ribosome‐binding deficient (ΔSΔCTD) S‐HA‐ZAKα constructs were treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed as in (B).

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