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: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. DIC image of rdLS during patch-clamp recording. Scale bar: 500 µm. b. Example traces of IPSCs before or 15 min after application of 300 nM stressin-1. c. Frequency of IPSCs. d. Amplitude of IPSCs. e. IPSCs area under the curve. For c-e, points are obtained from individual cells recorded from separate slices in 6 mice. f. C57BL/6J wild-type mice injected in rdLS with AAV2/1 Syn.GCaMP6f and implanted with an optical ferrule above rdLS. Implanted mice were presented with novel and familiar mice. g. Interaction time during social presentation. Dots are from 9 recording sessions using 5 mice. h. Average peak amplitude of the z-score during presentation of a novel or familiar mouse. Paired t test: p = 0.007. i. Frequency of events during presentation of a novel or a familiar mouse. Paired t test: p = 0.6. j. Discrimination index for social familiarity preference calculated from z-scores. One-sample t tests compared to 0: p = 0.006. k. Immunohistochemistry images of c-Fos labelling in rdLS following social presentation with a novel or familiar mouse (same experiment than ). Scale bars: 500 µm. l. Density of rdLS cells positive for c-Fos. We made one observation on each side of a rLS section. 4 mice per group. Unpaired t test, p = 0.0003. m. Density of rdLS cells positive for c-Fos vs. interaction time during social interaction. Each point represents one mouse. n. Percentage of layer 2/3 ILA CRH cells positive for c-Fos (cf. ) vs. density of rdLS cells positive for c-Fos following social interaction. Each point represents a mouse. o-p. Immunohistochemistry images of c-Fos labelling in ILA (o) and rdLS (p). CRH-Cre mice injected in ILA with AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(anti- Crh ) or AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled) were presented with a familiar mouse for 2 min before being processed for immunohistochemistry. Scale bars: 300 µm. q. Duration of interaction during familiar presentation. Each point is one mouse. Unpaired t test, p = 0.001. r. Percentage of layer 2/3 ILA CRH cells positive for c-Fos in layer 2/3 of ILA. Each point corresponds to each side of 2 sections. 9 mice per group. s. Density of rdLS cells positive for c-Fos. We made one observation on each side of a rLS section. 9 mice per group. Unpaired t test, p < 0.0001. t. Percentage of layer 2/3 ILA CRH cells positive for c-Fos vs. density of rdLS cells positive for c-Fos following social interaction with a familiar mouse. Each point represents one mouse. u. C57BL/6J wild-type mice were injected with AA2/2 hSyn1.hChR2(H134R)-mCherry or AA2/2 hSyn1.mCherry as control and an optical fiber was implanted above the injection site. Mice were then presented to a familiar mouse for 2 min meanwhile 450 nm light was applied (20 Hz, 1 ms). Mice were also run without light as additional controls. Scale bar: 1 mm. v. Total interaction time with familiar mouse. Each point represents one mouse. One-way ANOVA: F 3,32 = 7.05, p = 0.0009. Dunnett’s multiple comparisons tests: ChR-light vs. YFP-no light p = 0.0005, ChR-light vs. ChR-no light p = 0.006, ChR-light vs. YFP-light p = 0.01. w. Average duration of each bout of social interaction. Each point represents one mouse. One-way ANOVA: F 3,31 = 10.62, p < 0.0001. Dunnett’s multiple comparisons tests: ChR-light vs. YFP-no light p = 0.0001, ChR-light vs. ChR-no light p = 0.0001, ChR-light vs. YFP-light p = 0.002. x. Total distance travelled. Each point represents one mouse. y. Schematic of the vCA1-ILA CRH -rdLS circuit. For the entire figure, bar graphs represent mean ± S.E.M.

Article Snippet: After 10 min of stable baseline recording, stressin-1 (300 nM, Tocris # 1608) was applied following a 1:1000 dilution from stock solution into the ACSF.

Techniques: Patch Clamp, Injection, Immunohistochemistry, shRNA

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. In vitro whole-cell patch-clamp of rdLS neurons. a1. Example trace of IPSCs before or 15 min after application of ACSF. a2. Number of IPSCs. Points are individual cells recorded in 5 mice. a3. Frequency of IPSCs. a4. Amplitude of IPSCs. a5. IPSCs area under the curve. b. Neurons recorded in vLS before and after application of 300 nM stressin-1. b1. DIC image of the LS region where cells were recorded. Scale bar: 200 µm. b2. Example trace of IPSCs before or after 15 min 300 nM stressin-1. b3. Number of IPSCs. Points are individual cells recorded in 5 mice. b4. Frequency of IPSCs. b5. Amplitude of IPSCs. b6. IPSCs area under the curve. For the entire figure, bar graphs represent mean ± S.E.M.

Article Snippet: After 10 min of stable baseline recording, stressin-1 (300 nM, Tocris # 1608) was applied following a 1:1000 dilution from stock solution into the ACSF.

Techniques: In Vitro, Patch Clamp

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