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: Aging (Albany NY)

Article Title: miR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation

doi: 10.18632/aging.103834

Figure Lengend Snippet: Protein and mRNA expression of TULA-2, SYK Y323 and VEGFR-2 Y1175 in ECs. ECs were incubated under normal culture conditions. ( A ) RT-qPCR showed no significant change in the mRNA levels of TULA-2 in OECs. ( B ) Western blot analysis revealed an increase in the protein levels of TULA-2 in OECs, and the phosphorylation levels of SYK Y323 and VEGFR-2 Y1175 were decreased in OECs (*P < 0.05, n = 3).

Article Snippet: After blocking with TBST supplemented with 5% bovine serum albumin (BSA) (BioFroxx Inc, Germany), the PVDF membrane was incubated at 4°C overnight with primary antibodies against mouse TULA-2 (1:500; cat. no. sc-514612; Santa Cruz, CA, USA), SYK (1:1000; cat. no. 13198; Cell Signaling Technologies, Beverly, USA), p-SYK Y323 (1:1000; cat. no. 2715; Cell Signaling Technologies), VEGFR-2 (1:1000; cat. no. 9698; Cell Signaling Technologies), p-VEGFR-2 Y1175 (1:1000; cat. no. 2478; Cell Signaling Technologies) and GAPDH (1:1000; cat. no. 5174; Cell Signaling Technologies).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Phospho-proteomics

Journal: Aging (Albany NY)

Article Title: miR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation

doi: 10.18632/aging.103834

Figure Lengend Snippet: The miR-25-3p inhibitor negatively regulates the angiogenic signaling pathway by reducing angiogenic growth factor expression. ( A ) RT-qPCR showed no significant change in the TULA-2 mRNA level in YECs after transfection with the miR-25-3p inhibitor. ( B ) Western blot analysis showed that the miR-25-3p inhibitor upregulated the protein level of TULA-2 and downregulated the phosphorylation levels of SYK Y323 and VEGFR-2 Y1175 in YECs (n=3, data are expressed as the mean ± SEM, *P < 0.05 versus the negative control).

Article Snippet: After blocking with TBST supplemented with 5% bovine serum albumin (BSA) (BioFroxx Inc, Germany), the PVDF membrane was incubated at 4°C overnight with primary antibodies against mouse TULA-2 (1:500; cat. no. sc-514612; Santa Cruz, CA, USA), SYK (1:1000; cat. no. 13198; Cell Signaling Technologies, Beverly, USA), p-SYK Y323 (1:1000; cat. no. 2715; Cell Signaling Technologies), VEGFR-2 (1:1000; cat. no. 9698; Cell Signaling Technologies), p-VEGFR-2 Y1175 (1:1000; cat. no. 2478; Cell Signaling Technologies) and GAPDH (1:1000; cat. no. 5174; Cell Signaling Technologies).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Phospho-proteomics, Negative Control

Journal: Aging (Albany NY)

Article Title: miR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation

doi: 10.18632/aging.103834

Figure Lengend Snippet: MiR-25-3p mimics positively regulate the angiogenic signaling pathway by increasing angiogenic growth factor expression. ( A ) RT-qPCR showed no significant change in the TULA-2 mRNA level in OECs after transfection with miR-25-3p mimics. ( B ) Western blot analysis showed that miR-25-3p mimics downregulated the protein level of TULA-2 and upregulated the phosphorylation levels of SYK Y323 and VEGFR-2 Y1175 in OECs (n=3, data are expressed as the mean ± SEM, *P < 0.05 versus the negative control).

Article Snippet: After blocking with TBST supplemented with 5% bovine serum albumin (BSA) (BioFroxx Inc, Germany), the PVDF membrane was incubated at 4°C overnight with primary antibodies against mouse TULA-2 (1:500; cat. no. sc-514612; Santa Cruz, CA, USA), SYK (1:1000; cat. no. 13198; Cell Signaling Technologies, Beverly, USA), p-SYK Y323 (1:1000; cat. no. 2715; Cell Signaling Technologies), VEGFR-2 (1:1000; cat. no. 9698; Cell Signaling Technologies), p-VEGFR-2 Y1175 (1:1000; cat. no. 2478; Cell Signaling Technologies) and GAPDH (1:1000; cat. no. 5174; Cell Signaling Technologies).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Phospho-proteomics, Negative Control

Journal: Aging (Albany NY)

Article Title: miR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation

doi: 10.18632/aging.103834

Figure Lengend Snippet: TULA-2 knockdown positively regulates angiogenesis in vitro and increases p-SYK Y323 and p-VEGFR-2 Y1175 expression at the protein level. ( A ) Transwell assays showed that TULA-2 siRNA promoted the migration ability of OECs, and co-transfection of OECs with both si-TULA-2 and miR-25-3p inhibitors did not suppress the effect of TULA-2 upregulation on EC migration relative to the effects of siRNA-NC (n=5; Scale bar, 100 μm). ( B ) Tube formation determined on Matrigel showed that TULA-2 siRNA increased the total tube length, and co-transfection of OECs with both si-TULA-2 and miR-25-3p inhibitors did not suppress the effect of TULA-2 upregulation on EC tube formation relative to the effects of siRNA-NC (n=4; scale bar, 100 μm). ( C ) Western blot analysis of relative TULA-2, SYK Y323 and VEGFR-2 Y1175 expression in OECs transfected with the negative control, si-TULA-2, si-TULA-2+ miR-25-3p inhibitor and miR-25-3p inhibitor (data are expressed as the mean ± SEM, *P < 0.05, **P < 0.01 versus the negative control).

Article Snippet: After blocking with TBST supplemented with 5% bovine serum albumin (BSA) (BioFroxx Inc, Germany), the PVDF membrane was incubated at 4°C overnight with primary antibodies against mouse TULA-2 (1:500; cat. no. sc-514612; Santa Cruz, CA, USA), SYK (1:1000; cat. no. 13198; Cell Signaling Technologies, Beverly, USA), p-SYK Y323 (1:1000; cat. no. 2715; Cell Signaling Technologies), VEGFR-2 (1:1000; cat. no. 9698; Cell Signaling Technologies), p-VEGFR-2 Y1175 (1:1000; cat. no. 2478; Cell Signaling Technologies) and GAPDH (1:1000; cat. no. 5174; Cell Signaling Technologies).

Techniques: Knockdown, In Vitro, Expressing, Migration, Cotransfection, Western Blot, Transfection, Negative Control

Journal: Aging (Albany NY)

Article Title: miR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation

doi: 10.18632/aging.103834

Figure Lengend Snippet: The interaction of TULA-2 and SYK inhibits the phosphorylation of SYK Y323 and VEGFR-2 Y1175. ( A ) Western blot analysis of the Co-IP experiment showed that SYK immunoprecipitated with anti-TULA-2, SYK and TULA-2, which was visualized by Western blot. ( B ) Western blot analysis of relative TULA-2, SYK Y323 and VEGFR-2 Y1175 expression in YECs transfected with the control, BAY 61-3606 (SYK inhibitor), and BAY 61-3606 + si-TULA-2. The phosphorylation levels of SYK Y323 and VEGFR-2 Y1175 were decreased in YECs after transfection with BAY 61-3606 or BAY 61-3606 + si-TULA-2 (data are expressed as the mean ± SEM, *P < 0.05 versus the negative control).

Article Snippet: After blocking with TBST supplemented with 5% bovine serum albumin (BSA) (BioFroxx Inc, Germany), the PVDF membrane was incubated at 4°C overnight with primary antibodies against mouse TULA-2 (1:500; cat. no. sc-514612; Santa Cruz, CA, USA), SYK (1:1000; cat. no. 13198; Cell Signaling Technologies, Beverly, USA), p-SYK Y323 (1:1000; cat. no. 2715; Cell Signaling Technologies), VEGFR-2 (1:1000; cat. no. 9698; Cell Signaling Technologies), p-VEGFR-2 Y1175 (1:1000; cat. no. 2478; Cell Signaling Technologies) and GAPDH (1:1000; cat. no. 5174; Cell Signaling Technologies).

Techniques: Phospho-proteomics, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing, Transfection, Control, Negative Control

Journal: Aging (Albany NY)

Article Title: miR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation

doi: 10.18632/aging.103834

Figure Lengend Snippet: The proposed novel mechanism by which miR-25-3p affects angiogenesis in aged mice via the TULA-2/SYK/VEGFR-2 pathway. Aged ECs utilize miR-25-3p to target TULA-2 as a downstream effector to transduce signals, further downregulating and suppressing the phosphorylation of SYK Y323 and VEGFR-2 Y1175 and thus suppressing the angiogenesis pathway and inhibiting the proliferation, migration and tube formation abilities of aged ECs.

Article Snippet: After blocking with TBST supplemented with 5% bovine serum albumin (BSA) (BioFroxx Inc, Germany), the PVDF membrane was incubated at 4°C overnight with primary antibodies against mouse TULA-2 (1:500; cat. no. sc-514612; Santa Cruz, CA, USA), SYK (1:1000; cat. no. 13198; Cell Signaling Technologies, Beverly, USA), p-SYK Y323 (1:1000; cat. no. 2715; Cell Signaling Technologies), VEGFR-2 (1:1000; cat. no. 9698; Cell Signaling Technologies), p-VEGFR-2 Y1175 (1:1000; cat. no. 2478; Cell Signaling Technologies) and GAPDH (1:1000; cat. no. 5174; Cell Signaling Technologies).

Techniques: Transduction, Phospho-proteomics, Migration

Journal: Cell reports

Article Title: Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells

doi: 10.1016/j.celrep.2018.06.054

Figure Lengend Snippet: (A) Left: cartoon showing a cell culture assay in which PD-1 transduced Jurkat cells were stimulated with three types of antigen-loaded Raji B cells: (1) parental Raji cells that express neither PD-L1 nor PD-1, (2) Raji cells transduced with only PD-L1 (PD-L1+), and (3) Raji transduced with both PD-L1 and PD-1 (PD-L1+/PD-1+). On the right are FACS histograms showing PD-1 and PD-L1 surface expression of parental Jurkat cells, PD-1–mGFP transduced Jurkat (PD-1+) cells, parental Raji cells, PD-L1–mCherry transduced Raji (PD-L1+) cells, and Raji cells co-transduced with both PD-1–mGFP and PD-L1–mCherry (PD-L1+/PD-1+). a.u., arbitrary units. (B–D) Jurkat cells expressing PD-1–mGFP (PD-1+) were conjugated with Raji B cells (parental) (B), Raji cells transduced with only PD-L1–mCherry (PD-L1+) (C), or Raji cells co-transduced with PD-L1–mCherry and unlabeled PD-1 (PD-L1+/PD-1+) (D), as illustrated in the cartoon on the left (CD28 and B7 are omitted in this cartoon for simplicity). Shown on the right are confocal images of the cell conjugate acquired 2 min after cell-cell contact. mGFP and mCherry signals are shown as green and magenta, respectively. Scale bars, 10 µm. (E) Bar graph summarizing the interface enrichment indices (calculated as described in STAR Methods) of the three conditions shown in (B). Data are shown as mean ± SEM; n = 35 cells from three independent experiments. See also . (F) A representative western blot showing the levels ZAP70-Y493 phosphorylation and p85 (PI3K regulatory subunit) co-immunoprecipitated (IP) with CD28 from the lysates of the indicated Jurkat-Raji co-culture. Jurkat cells expressing PD-1–mGFP (PD-1+) were stimulated with Raji cells (parental), Raji transduced with PD-L1–mCherry (PD-L1+), or Raji cells co-transduced with PD-L1–mCherry and PD-1–mGFP (PD-L1+/PD-1+); the times at which the co-culture was lysed are indicated (STAR Methods). WCL, whole cell lysate. (G and H) Bar graphs summarizing immunoblots in (F), including pY493-ZAP70 immunoblot (G) and CD28 coIP p85 immunoblot (H). The optical density corresponding to each band was quantified by ImageJ and normalized to the conditions in which parental Raji cells were used as the APCs. Data are presented as mean ± SEM from three independent replicates. (I) Bar graph summarizing IL-2 levels in the medium of the indicated Jurkat-Raji co-culture, as described in (B)–(D), 24 hr after cell-cell contact. Data are presented as mean ± SEM from three independent measurements, with each run performed in triplicate.

Article Snippet: ZAP70 pY493 antibody , Cell Signaling Technology , Cat # 2704S; RRID: AB_2217457.

Techniques: Cell Culture, Transduction, Expressing, Western Blot, Phospho-proteomics, Immunoprecipitation, Co-Culture Assay

Journal: Cell reports

Article Title: Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells

doi: 10.1016/j.celrep.2018.06.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ZAP70 pY493 antibody , Cell Signaling Technology , Cat # 2704S; RRID: AB_2217457.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Software, Imaging