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

Journal: The Journal of biological chemistry

Article Title: Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates.

doi: 10.1074/jbc.273.43.28332

Figure Lengend Snippet: FIG. 2. The SH2 domains of SHP-1 and SHP-2 mediate binding to PECAM-1-(658–668) Tyr(P)-663 and PECAM-1-(681–691) Tyr(P)-686 peptides. A, 2 mg of each recombinant fusion protein encompassing either GST alone (lanes 1, 3, 5, and 7) or the GST containing amino- and carboxyl-terminal SH2 domains of SHP-1 (lanes 2, 4, 6, and 8) were incubated with 10 mg of biotinylated phosphorylated and nonphosphorylated versions of PECAM-1-(658–668) and PECAM- 1-(681–691) peptides. Bound protein-peptide complexes were recovered using streptavidin-agarose beads, separated by SDS-PAGE, and probed with a polyclonal antibody directed to the N-terminal SH2 domains of SHP-1. Note that the PECAM-1-(658–668) Tyr(P)-663 and PECAM-1- (681–691) Tyr(P)-686 peptides bound avidly to the SH2 domains of SHP-1 (lanes 4 and 8). B, experimental conditions as in A, except the amino- and carboxyl-terminal SH2 domains of SHP-1 were substituted for SHP-2, and immunoblot analysis was probed with a polyclonal antibody directed to the amino-terminal SH2 domains of SHP-2. Note that the PECAM-1-(658–668) Tyr(P)-663 and PECAM-1-(681–691) Tyr(P)-686 peptides bound avidly to the SH2 domains of SHP-2 (lanes 4 and 8). The positions of the molecular mass standards are shown and expressed in kilodaltons, and the positions of GST-N-SH2-C-SH2- SHP-1 and GST-N-SH2-C-SH2-SHP-2 are indicated.

Article Snippet: A polyclonal antibody directed to the NH2- and COOH-terminal SH2 domains of SHP-2 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Binding Assay, Recombinant, Incubation, SDS Page, Western Blot

Journal: eLife

Article Title: m6A modifications regulate intestinal immunity and rotavirus infection

doi: 10.7554/elife.73628

Figure Lengend Snippet: Figure 4. Rotavirus suppresses ALKBH5 expression through NSP1 to evade immune defense. (a) WT mice were infected by RV EW at 8 days post birth. Immunoblotting with antibodies target ALKBH5, FTO, METTL14 and METTL3 in ileum tissue from mice infected with RV EW at two dpi or treated with PBS. (b) Quantitative analysis of (a) (mean ± SEM), Statistical significance was determined by Student’s t-test (*p < 0.05). (c–e) Alkbh5ΔIEC mice and littermate controls were infected by RV EW at 8 days post birth. qPCR analysis of indicated genes expression in ileum (c), viral shedding in feces (d), and viral proteins expression in ileum (e), from Alkbh5ΔIEC mice or littermate controls at 4 days post infection (littermate WT n = 6, Alkbh5ΔIEC n = 5, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, NS., not significant). (f) Immunoblotting with antibodies target ALKBH5, NSP1, VP6 and GAPDH in HEK293 cells infected by SA11-4F and SA11-NSP1null (MOI = 1) for 24 hr. (g) Graphical abstract

Article Snippet: METTL3 (abcam, ab195352, 1:2000), METTL14 (sigma, HPA038002, 1:2000), ALKBH5 (sigma, HPA007196, 1:2000), FTO (abcam, ab92821), NSP1 and VP6 (gift from Harry B. Greenberg lab), GAPDH (proteintech), TUBULIN (proteintech), beta- ACTIN (proteintech), Phospho- IRF- 7 (Ser437/438) (D6M2I) (CST), Phospho- TBK1/NAK (Ser172) (D52C2) (CST), TBK1/NAK (D1B4) (CST), and IRF- 7 (D8V1J) (CST) antibodies were used in accordance with the manufacturer’s instructions.

Techniques: Expressing, Infection, Western Blot

Journal: PLOS ONE

Article Title: Short communication: Upregulation of hypoxia/reoxygenation-induced Shc3 by downregulated miR-455-5p, suppresses trophoblast invasion and is associated with placental inflammation and angiogenesis in preeclampsia

doi: 10.1371/journal.pone.0314544

Figure Lengend Snippet: (A) Heatmap of RNA-Seqs. (B) Selection of potential miR-455-5p downstream genes. (C) Schematic diagram of luciferase reporter-Shc3 3’UTR constructs. (D) The outcomes of miR-455-5p overexpression on the luciferase activity in HTR8/SVneo cells transfected with the WT or MUT luciferase reporter constructs. (E) The effect of miR-455-5p inhibition and overexpression on Shc3 mRNA expression in HTR8/SVneo cells. (F) The level of Shc3 protein expression after miR-455-5p inhibition and overexpression in HTR8/SVneo. Data presented shows the average values ± standard deviation (**p < 0.01, *p < 0.05). The findings presented were obtained from a minimum of three independent experiments.

Article Snippet: SHC3, Beta Actin antibodies and HRP-conjugated Affinipure Goat Anti Rabbit IgG(H+L) were obtained from Proteintech (Wuhan, China).

Techniques: Selection, Luciferase, Construct, Over Expression, Activity Assay, Transfection, Inhibition, Expressing, Standard Deviation

Journal: PLOS ONE

Article Title: Short communication: Upregulation of hypoxia/reoxygenation-induced Shc3 by downregulated miR-455-5p, suppresses trophoblast invasion and is associated with placental inflammation and angiogenesis in preeclampsia

doi: 10.1371/journal.pone.0314544

Figure Lengend Snippet: (A) The migration and invasion of HTR8/SVneo cells with miR-455-5p overexpression plasmids co-cultured with the plasmids of Shc3, or Shc3-NC in H/R. (B) Flow cytometry data of the apoptosis of HTR8/SVneo cells with miR-455-5p overexpression plasmids co-cultured with the plasmids expressing Shc3 or Shc3-NC in H/R. (C) Western blot results of Shc3 expression in HTR8/SVneo cells transfected with the miR-455-5p mimic, Shc3, or Shc3-NC. (D) The results of Shc3 expression examined by immunohistochemistry. Data presented indicate the average values ± standard deviation (**p < 0.01, *p < 0.05). The findings presented were obtained from a minimum of three independent experiments.

Article Snippet: SHC3, Beta Actin antibodies and HRP-conjugated Affinipure Goat Anti Rabbit IgG(H+L) were obtained from Proteintech (Wuhan, China).

Techniques: Migration, Over Expression, Cell Culture, Flow Cytometry, Expressing, Western Blot, Transfection, Immunohistochemistry, Standard Deviation

Journal: PLOS ONE

Article Title: Short communication: Upregulation of hypoxia/reoxygenation-induced Shc3 by downregulated miR-455-5p, suppresses trophoblast invasion and is associated with placental inflammation and angiogenesis in preeclampsia

doi: 10.1371/journal.pone.0314544

Figure Lengend Snippet: (A~B). H/R promotes the production of Shc3, which may be transported to the extracellular space through medium/large extracellular vesicles. (A) Western blot results of Shc3 expression in hypoxia, H/R, and normoxia, respectively. (B) Volcano plot of Shc3 mRNA expression in placental tissue, extracellular medium/large vesicles and small vesicles. (C~E). Shc3 is involved in placental inflammation and angiogenesis inhibition. (C) Hematoxylin-eosin staining results on normotensive placental tissues and high Shc3 expression PE placental tissues. (D) The level of Shc3 in EA.hy926 after transfection with the plasmids of Shc3, and Shc3-NC, in H/R compared with controls. (E) Angiogenesis of EA.hy926 after transfection with the plasmids of Shc3, and Shc3-NC in H/R, as determined by the number of master junction and master segment length.

Article Snippet: SHC3, Beta Actin antibodies and HRP-conjugated Affinipure Goat Anti Rabbit IgG(H+L) were obtained from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Inhibition, Staining, Transfection