WNK1 is not required for c-MYC upregulation and is not activated by PI3K, AKT, IKK2, or MEK downstream of CD40. (A and B) Immunoblot analysis (top) of cell lysates from control or WNK1-deficient B cells stimulated with anti-IgM (A) or CD40L (B) for the indicated times, probed with antibodies to MYC or α-TUBULIN. Graphs (below) show mean ± SEM levels of MYC normalized to the abundance of α-TUBULIN in each lane. (C) Immunoblot analysis (top) of cell lysates from WT mouse B cells stimulated with LPS for the indicated times, probed with antibodies to p-OXSR1 and α-TUBULIN. Graph shows mean ± SEM levels of p-OXSR1 normalized to the abundance of α-TUBULIN in each lane. (D and E) Top: Immunoblots of total cell lysates from WT mouse B cells treated with vehicle (DMSO), a PI3K inhibitor (PI-103), or an AKT inhibitor (MK2206; D), an IKK2 inhibitor (BI605906) or a MEK1 and MEK2 inhibitor (PD0325901; E) and stimulated for the indicated times with CD40L, probed with antibodies to p-OXSR1, p-AKT, p-PRAS40, p-p105, p-ERK2, or ERK2. Below: graphs of mean ± SEM abundance of p-OXSR1, p-AKT, p-PRAS40, p-p105, and pERK2 in the lanes above, normalized to ERK2. Two-way ANOVA (A, B, D, and E); Mann–Whitney test (C); *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001; ****, P < 0.0001. Sample sizes: six (A, B, D, and E); and five (C). Data are pooled from two (A and B) or three (C–E) independent experiments. Source data are available for this figure: SourceData FS4.