syk inhibitor bay61 3606  (MedChemExpress)

Journal: Nature immunology

Article Title: Molecular basis for potent B cell responses to antigen displayed on particles of viral size

doi: 10.1038/s41590-023-01597-9

Figure Lengend Snippet: a,b, Pooled splenocytes and lymphocytes from MD4 mice were preincubated at 37 °C for 15 min with Rigel R568 IRAK1/IRAK4 inhibitor (IRAK1/IRAK4i; 1 μM), subsequently incubated for 20 min with the indicated stimuli (2.5 μM CpG and 1 pM pHELD), and then fixed, permeabilized and stained to detect pErk or pS6 as well as B220. Representative histograms depict pErk (a) or pS6 (b) in B220+ B cells. The graph depicts pErk MFI from three independent experiments. Data in b are representative of at least four independent experiments. Data were compared by unpaired two-tailed parametric t-test. c,d, Pooled splenocytes and lymphocytes from MD4 Myd88+/+ and MD4 Myd88−/− mice were stimulated with the indicated stimuli for 20 min and stained to detect pErk or pS6 in B220+ B cells as in a and b (10 μg ml−1 lipopolysaccharide (LPS), 2.5 μM CpG, 10 μg ml−1 anti-IgM, 1 μg ml−1 anti-CD40, 1 μg ml−1 sHEL-WT or 1 pM pHELT-HD (ED of 256)). Histograms depict pErk (c) or pS6 (d) and are representative of four (c) or three (d) independent experiments. e,f, As in a except with pretreatment with SYK inhibitor (SYKi; 1 μM Bay 61–3606) or BTK inhibitor (BTKi; 100 nM ibrutinib), followed by a 20-min incubation with the indicated stimuli: 1 pM and 0.1 pM (f) pHELD-HD (ED of 286), 1 μg ml−1 sHELD, 10 μg ml−1 anti-IgM and 2.5 μM CpG. Histograms are representative of four independent experiments. g,h, As in a except pretreatment with PI3K inhibitor (PI3Ki; 10 μM Ly290049) and stimuli (1 pM pHELT-HD ED = 256 in g; 1 pM pHELD-LD ED = 53 and pHELT-LD ED = 58 in h). Histograms are representative of four independent experiments.

Article Snippet: SYK inhibitor (Bay 61–3606, 1 μM) and PI3K inhibitor ( Ly290049 , 10 μM) were from Calbiochem, BTK inhibitor (ibrutinib, 100 nM) was from J. Taunton (University of California, San Francisco) and IRAK1/IRAK4 inhibitor (R568, 1 μM) was a gift from Rigel.

Techniques: Incubation, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Molecular basis for potent B cell responses to antigen displayed on particles of viral size

doi: 10.1038/s41590-023-01597-9

Figure Lengend Snippet: a. Signalosome assembly in naïve B cells upon BCR stimulation by soluble Ag. BCR signal transduction requires sequential action of Src family kinases (SFKs) and SYK kinase. CD19 engagement amplifies PI3K activation and production of PI(3,4,5)P3. While multiple SFKs can mediate ITAM signaling downstream of the BCR, the SFK LYN plays a non-redundant role in phosphorylating ITIM-containing inhibitory coreceptors which in turn recruit PTPases SHP1 and SHIP1 that suppress PIP3. Dynamic regulation of PIP3 at the plasma membrane controls amplitude of signaling by recruiting downstream mediators including AKT, BTK, and PLCγ2 to orchestrate transcriptional programs mediated by NFAT, NF-κB and other factors. b. SVLS with appropriately spaced epitopes robustly engage pre-existing BCR nanoclusters but evade co-inhibitory receptors and results in downstream signal amplification. SVLS do not rely upon CD19 engagement for signal amplification in vitro. In the absence of inhibitory PTPase engagement via ITIM-containing inhibitory receptors, PIP3 accumulates at the plasma membrane leading to enhanced and prolonged signalosome assembly and activity downstream of SVLS stimulation. Robust NFAT and NF-κB accumulation in the nucleus and AKT-dependent signals mimic co-stimulation and promote MYC expression, resulting in T-independent cell growth, survival, and proliferation.

Article Snippet: SYK inhibitor (Bay 61–3606, 1 μM) and PI3K inhibitor ( Ly290049 , 10 μM) were from Calbiochem, BTK inhibitor (ibrutinib, 100 nM) was from J. Taunton (University of California, San Francisco) and IRAK1/IRAK4 inhibitor (R568, 1 μM) was a gift from Rigel.

Techniques: Transduction, Activation Assay, Membrane, Amplification, In Vitro, Activity Assay, Expressing