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, 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, Inhibition, Immunoprecipitation

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