Journal: Cell Death & Disease

Article Title: Targeting SYK of monocyte-derived macrophages regulates liver fibrosis via crosstalking with Erk/Hif1α and remodeling liver inflammatory environment

doi: 10.1038/s41419-021-04403-2

Figure Lengend Snippet: HIF1α A protein and B mRNA levels were measured after CCL4 injection for 4 weeks and 8 weeks by Western Blot or PCR; C The correlation analysis of the expressions of SYK and HIF1α in CCL4 models (** p < 0.01); D The hepatocytes and macrophages expression of Hif1α in siCtrl and siSYK fibrotic livers. E And, 4 h before TAA injection, mice were injected with siCtrl and siSYK through the tail vein, and determine the transcription of Hif1α in the liver 1, 3, and 7 days after TAA injection Level (* p < 0.05); F Transfect mouse BMDM with shSYK lentivirus, then stimulate BMDM with LPS for 6 h, detect the protein levels of p-SYK, p-Erk1/2, HIF1α in macrophages; G The protein level of HIF1α in BMDM in the presence or absence of siSYK or Erk1/2 inhibitor Ravox.

Article Snippet: Use for SYK (Cell Signaling Technology, 13198), p-SYK (Bioss, bs-3434R), Erk1 + Erk2 (abcam, ab184699), p-Erk1 + p-Erk2 (abcam, ab214036), HIF1α (Cell Signaling Technology, 36169), TGF-β (abcam, ab215715), α-SMA (Cell Signaling Technology, 19245), β-actin (Cell Signaling Technology, 4970) antibody for detection, using chemiluminescence-based method for color development.

Techniques: Injection, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Functional characterization of T-cells from palatine tonsils in patients with chronic tonsillitis

doi: 10.1371/journal.pone.0183214

Figure Lengend Snippet: (A) CD4/CD8 T-cells were isolated from tonsils and resuspended in FCS-free medium. The total CD4(+)CD8(+) T-cell preparations were challenged with soluble or immobilized anti CD3/CD28 Abs to activate the TCR. Cells were lysed and cellular extracts were processed for Western Blot detection of total and phosphorylated versions of PLCγ, ZAP70, Erk and Akt. (B) Bands were quantified by densitometry and the ratio of phosphorylated to total protein was plotted as as fold activation of unstimulated samples (0 min). The quantification includes all measured tonsil samples. Note that the values for PTA-abs/blo and PTA-heal/blo represent perforce the same data and are plotted twice for illustrative reasons. Data are presented as mean + SEM. pPLCγ, pErk, pAkt and pZAP-70 mark the phosphorylated versions of the respective proteins. CT = chronic tonsillitis; PTA = peritonsillar abscess; HY = tonsillar hyperplasia; ton = tonsil; blo = blood; abs = abscess; hea = healthy.

Article Snippet: Antibodies for Western blot analysis: Akt (pan) Rabbit mAb #4685, LAT antibody #9166, p44/42 MAPK (Erk1/2) Rabbit mAb #4695, Phospho-Akt (Ser473) (D9E) XP Rabbit mAb #4060, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370, Phospho- LAT (Tyr191) antibody #3584, Phospho-PLCγ1 (Tyr783) antibody #2821 and Phospho-Zap70 (Tyr319)/Syk (Tyr352) antibody #2701 from Cell Signaling Technology (Cambridge, Great Britain).

Techniques: Isolation, Western Blot, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

doi: 10.1016/j.jbc.2022.101857

Figure Lengend Snippet: Syk binds to specific tyrosine residues of SCIMP via its dual SH2 domains. A , schematic diagram of Syk domains and its truncation constructs representing the two tandem SH2 domains with or without linker regions. B , GST-Syk-N-SH2 and Syk-C-SH2 proteins were used to pull down SCIMP from LPS-induced primary BMMs and immunoblotted with SCIMP antibody. C , GST-Syk-N-SH2 and Syk-C-SH2 proteins were used to pull down V5-SCIMP in SCIMP-deficient RAW264.7 cell lines reconstituted with wild type WT, Y58F, Y96F, or Y120F V5-SCIMP. D , coimmunoprecipitation of V5-SCIMP WT and Y58F, Y96F, or Y120F V5-SCIMP from RAW264.7 cell lysates with a V5 antibody, followed by immunoblotting for Syk. Panels B–D are representative of three independent experiments. BMMs, bone marrow–derived macrophages; LPS, lipopolysaccharides; SH2, Src homology domain 2; Syk, Spleen tyrosine kinase.

Article Snippet: Codon-optimized mouse TLR4-TIR (residues 670-835) gene was purchased from Genscript USA and was also subcloned in to the pGEX6p-1 vector. pGEX plasmids for GST-tagged Syk-N-SH2 (residues 6-114, #46520), Syk-C-SH2 (residues 148-264, #46519), and Syk-NC-SH2 (residues 6-264, #46521) domains were all purchased from Addgene.

Techniques: Construct, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

doi: 10.1016/j.jbc.2022.101857

Figure Lengend Snippet: Model of Syk recruitment by SCIMP to TLRs. The transmembrane adapter SCIMP constitutively binds Lyn via a PRD-SH3 interaction. Upon ligand activation of TLR4, activated Lyn kinase phosphorylates SCIMP at three tyrosine sites. The Y96 and Y120 residues then function as two docking sites for the tandem SH2 domains of Syk, which triggers a conformational change of Syk to expose its kinase domain for amplifying SCIMP and TLR4 phosphorylation and enhancing their interaction. SCIMP-scaffolded Syk helps to propagate TLR4 signal transduction to drive proinflammatory cytokine secretion. Syk, Spleen tyrosine kinase; TLRs, Toll-like receptors; SH2, Src homology 2.

Article Snippet: Codon-optimized mouse TLR4-TIR (residues 670-835) gene was purchased from Genscript USA and was also subcloned in to the pGEX6p-1 vector. pGEX plasmids for GST-tagged Syk-N-SH2 (residues 6-114, #46520), Syk-C-SH2 (residues 148-264, #46519), and Syk-NC-SH2 (residues 6-264, #46521) domains were all purchased from Addgene.

Techniques: Activation Assay, Transduction