Journal: The Journal of Experimental Medicine

Article Title: B cell–intrinsic requirement for WNK1 kinase in antibody responses in mice

doi: 10.1084/jem.20211827

Figure Lengend Snippet: Signaling from the BCR and CXCR5 activates WNK1 via PI3K and AKT. (A–J) Top: Immunoblots of cell lysates from mouse B cells stimulated for the indicated times with anti-IgM (A–D and I) or CXCL13 (E–H and J) using WT (A and E), WNK1-deficient or control B cells (B and F), B cells expressing kinase-inactive WNK1-D368A or control B cells (C and G), or WT B cells treated with vehicle (DMSO), an inhibitor of WNK family kinases (WNK463; D and H), a PI3K inhibitor (PI-103) or an AKT inhibitor (MK2206; I and J), probed with antibodies to phosphorylated OXSR1 (p-OXSR1), α-TUBULIN, phosphorylated AKT (p-AKT), phosphorylated PRAS40 (p-PRAS40) or ERK2. Bottom: Mean ± SEM abundance of p-OXSR1, p-AKT, and p-PRAS40 in the lanes above, normalized to α-TUBULIN or ERK2. Mann–Whitney test (A and E), two-way ANOVA (B–D, F–H, I, and J); *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001; ****, P < 0.0001. Sample sizes: four (B, E, and J); five (C, F, G, and I); seven (D and H); and eight (A). Data are pooled from two (B, C, E, F, G, I, and J) or three (A, D, and H) independent experiments. Source data are available for this figure: .

Article Snippet: The following antibodies and reagents were used for the detection of proteins by immunoblotting: anti-pS325-OXSR1/pS383-STK39 (MRC-PPU), anti-OXSR1 (MRC-PPU) anti-α-TUBULIN (TAT-1, Cell Services STP, The Francis Crick Institute), anti-pS473-AKT (193H12; Cell Signaling Technology), anti-pT246-PRAS40 (C77D7; Cell Signaling Technology), anti-ERK2 (D-2; Santa Cruz Biotechnology), anti-SYK (5F5; BioLegend), anti-pS932-p105 (18E6; Cell Signaling Technology), anti-pT202/pT204-ERK1/2 (197G2; Cell Signaling Technology), and anti-MYC (Y69; Abcam).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: B cell–intrinsic requirement for WNK1 kinase in antibody responses in mice

doi: 10.1084/jem.20211827

Figure Lengend Snippet: 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: .

Article Snippet: The following antibodies and reagents were used for the detection of proteins by immunoblotting: anti-pS325-OXSR1/pS383-STK39 (MRC-PPU), anti-OXSR1 (MRC-PPU) anti-α-TUBULIN (TAT-1, Cell Services STP, The Francis Crick Institute), anti-pS473-AKT (193H12; Cell Signaling Technology), anti-pT246-PRAS40 (C77D7; Cell Signaling Technology), anti-ERK2 (D-2; Santa Cruz Biotechnology), anti-SYK (5F5; BioLegend), anti-pS932-p105 (18E6; Cell Signaling Technology), anti-pT202/pT204-ERK1/2 (197G2; Cell Signaling Technology), and anti-MYC (Y69; Abcam).

Techniques: Western Blot, MANN-WHITNEY