Journal: The Journal of Biological Chemistry

Article Title: Bruton’s tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids

doi: 10.1016/j.jbc.2022.102231

Figure Lengend Snippet: G3BP1 is tyrosine phosphorylated upon p(I:C) stimulation and is inhibited by prior treatment with BTK inhibitors. A , bone-marrow derived macrophages (BMDMs) were stimulated with p(I:C) for 2 and 4 h (h), and whole cell lysates (WCLs) were immunoprecipitated (IP) with an anti-G3BP1 antibody and immunoblotted (IB) with 4G10 antibody to examine tyrosine phosphorylation of endogenous G3BP1. B , HeLa cells were stimulated with p(I:C) for 2 h, and endogenous G3BP1 was IP and IB with 4G10 antibody to examine for tyrosine phosphorylation. C , HEK293T cells were transfected with HA-tagged G3BP1 and stimulated with p(I:C) for 2 h. WCL was IP with an anti-HA antibody and IB with 4G10 antibody. D , HEK293T cells bearing HA-tagged G3BP1 were nontreated or pretreated with LFM-A13 or terreic acid prior to p(I:C) stimulation, and G3BP1 was IP from WCL with anti-HA and IB with 4G10 antibodies. Anti-GAPDH IB served as loading controls. BTK, Bruton’s tyrosine kinase; G3BP1, RAS-GTPase-activating protein (SH3 domain)-binding protein 1; p(I:C), polyinosinic:polycytidylic acid.

Article Snippet: BMDM, HEK293T, and LN-229 cells were pretreated with LFM-A13, terreic acid (MedChem Express), and PP2 (Millipore) for an hour prior to stimulation with various ligands.

Techniques: Derivative Assay, Immunoprecipitation, Phospho-proteomics, Transfection, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Bruton’s tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids

doi: 10.1016/j.jbc.2022.102231

Figure Lengend Snippet: BTK binds and phosphorylates G3BP1 upon p(I:C) stimulation . A , confocal microscopy study of G3BP1 and BTK colocalization. HeLa cells were transfected with HA-tagged G3BP1 and FLAG-tagged BTK and 24 h later, left untreated (upper panel) or stimulated with p(I:C) (lower panel). HA- and FLAG-tagged proteins were visualized using fluorochrome-conjugated antibodies (green for G3BP1 and red for BTK). B , enhanced direct binding of overexpressed BTK and G3BP1. HEK293T cells were transfected with FLAG-tagged BTK and HA-tagged G3BP1, and WCLs were IP and IB with relevant antibodies as indicated to examine BTK-G3BP1 interaction or IB with anti-FLAG or anti-HA antibodies to examine transfection efficiency and protein expression of individual constructs. C , G3BP1 is tyrosine phosphorylated in the presence of BTK co-expression. HEK293T cells were transfected with FLAG-tagged BTK and HA-tagged G3BP1, and WCLs were IP with anti-HA and IB with 4G10 antibodies to examine for tyrosine phosphorylation of G3BP1. D , BTK inhibition attenuated G3BP1 binding and tyrosine phosphorylation. HEK293T cells bearing HA-tagged G3BP1 and FLAG-tagged BTK were untreated or stimulated with p(I:C) in the absence or presence of the BTK inhibitor LFM-A13, and G3BP1 was IP from WCL with anti-HA antibody and probed with anti-FLAG antibody to examine BTK-G3BP1 binding and with 4G10 antibody to assess G3BP1 tyrosine phosphorylation. BTK, Bruton’s tyrosine kinase; G3BP1, RAS-GTPase-activating protein (SH3 domain)-binding protein 1; IB, immunoblotted; IP, immunoprecipitated; p(I:C), polyinosinic:polycytidylic acid; WCL, whole cell lysate.

Article Snippet: BMDM, HEK293T, and LN-229 cells were pretreated with LFM-A13, terreic acid (MedChem Express), and PP2 (Millipore) for an hour prior to stimulation with various ligands.

Techniques: Confocal Microscopy, Transfection, Binding Assay, Expressing, Construct, Phospho-proteomics, Inhibition, Immunoprecipitation

Journal: Virulence

Article Title: Staphylococcus aureus toxins mediate endothelial Thrombomodulin release during severe invasive infections

doi: 10.1080/21505594.2025.2605767

Figure Lengend Snippet: Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum MMP inhibitor Marimastat (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.

Article Snippet: The experimental conditions included the following treatments: (i) negative control, (ii) S. aureus overnight culture supernatants from strains M37 or P08 (7,000 ng/mL protein), (iii) 50 ng/mL recombinant α-toxin (IBT Bioservices, 1401–002), and (iv) co-treatment with 1300 nM broad-spectrum MMP inhibitor Marimastat (Tocris Bioscience, 2631; CAS: 154039-60–8) or 10,6 μM ADAM10 inhibitor GI254023X (Sigma-Aldrich, SML0789) in combination with S. aureus supernatants or recombinant α-toxin.

Techniques: Isolation, Cell Culture, Expressing, Lysis, Western Blot, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F

doi: 10.1111/jcmm.12005

Figure Lengend Snippet: Effects of the different compounds used in this study

Article Snippet: LFM-A13 was purchased from Axxora (San Diego, CA, USA), Sunitinib was from Cayman Chemical Company (Ann Arbor, MI, USA) and TG101348 was also provided by Axon Medchem (Groningen, The Netherlands).

Techniques: Gene Assay, Translocation Assay, Proliferation Assay, Inhibition