Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated

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 immunology (Baltimore, Md. : 1950)

Article Title: The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain.

doi: 10.4049/jimmunol.1401299

Figure Lengend Snippet: FIGURE 2. AnxA5 and AnxA13 translocate to the cell surface upon induction of apoptosis. (A) Flow cytometric analysis of aS2. Sixteen hours after UV-C irradiation (250 mJ/cm2), mock- or AnxA1/5/13-transfected aS2 cells were early apoptotic (AnxV+/7-AAD2) and stained using biotin- conjugated anti-6xHis streptavidin-allophycocyanin. (B) Immunoblot analysis of cytosolic and membrane (EDTA wash) fractions of viable (0 h) or early UV-C–irradiated apoptotic CEM cells (50 mJ/cm2, 2–6 h). (C) Quantification of cell viability of UV-C–irradiated early apoptotic CEM cells in (B) by flow cytometry using AnxV-FITC/7-AAD.

Article Snippet: Commercially available Abs for detection of MAPK, a-tubulin, and b-actin were purchased as follows: AnxA5 (AF399; R&D Systems), AnxA13 (AF4149; R&D Systems), p-ERK (E10; Cell Signaling), ERK1 (MK12; BD), p-JNK (G9; Cell Signaling), JNK1/3 (C17; Santa Cruz Biotechnology), p-p38 (D3F9; Cell Signaling), p38a (5F11; Cell Signaling), a-tubulin (B-5-1-2; Sigma-Aldrich), and b-actin (AC-15; Abcam).

Techniques: Irradiation, Transfection, Staining, Western Blot, Membrane, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain.

doi: 10.4049/jimmunol.1401299

Figure Lengend Snippet: FIGURE 3. AnxA5 and AnxA13 inhibit proinflammatory cytokine secretion of BMDCs upon TLR stimulation. (A) AnxA1, AnxA5, and AnxA13. The N-terminal domain is unique for each Anx family member and is depicted in yellow. The highly conserved core domain is shown in blue. (B–D) GM-CSF– differentiated BMDCs from Tlr42/2 mice were incubated with the indicated concentrations of recombinant protein. Six to eight hours later, cells were stimulated with 40 nM CpG o/n. TNF-a (B), IL-12p40 (C), or IL-6 (D) concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of three independent experiments. (E and F) GM-CSF–differentiated BMDCs from Tlr42/2 mice were incubated with the indicated ratio of apoptotic Jurkat T cells (aJ) or aS2 AnxA1, aS2 AnxA5, or aS2 mock. Four to six hours later, cells were stimulated with 40 nM CpG o/n. TNF-a concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of four independent experiments. (G and H) Flt3L-differentiated BMDCs from Tlr42/2 mice were incubated with the indicated concentrations of recombinant protein. Six to eight hours later, cells were stimulated with 40 nM CpG o/n. TNF-a (G) or IL-10 (H) concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

Article Snippet: Commercially available Abs for detection of MAPK, a-tubulin, and b-actin were purchased as follows: AnxA5 (AF399; R&D Systems), AnxA13 (AF4149; R&D Systems), p-ERK (E10; Cell Signaling), ERK1 (MK12; BD), p-JNK (G9; Cell Signaling), JNK1/3 (C17; Santa Cruz Biotechnology), p-p38 (D3F9; Cell Signaling), p38a (5F11; Cell Signaling), a-tubulin (B-5-1-2; Sigma-Aldrich), and b-actin (AC-15; Abcam).

Techniques: Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain.

doi: 10.4049/jimmunol.1401299

Figure Lengend Snippet: FIGURE 4. AnxA5 and AnxA13 specifically downregulate the expression of costimulatory molecules on BMDCs. (A–G) GM-CSF–differentiated BMDCs from Tlr42/2 mice were incubated with 500 nM of the indicated recombinant protein. Six to eight hours later, cells were stimulated with 20 nM CpG. After 2 d, cells were stained with Abs against MHC I (A), MHC class II (B), CD40 (C), CD80 (D), CD86 (E), PD-L1 (F), and PD-L2 (G). Depicted are the mean fluorescence intensity (MFI) of individual mice and the group mean. (H) Quantification of cell viability of BMDCs of mouse 1 used in (A)–(G) by flow cytometry using AnxV-FITC/7-AAD. *p , 0.05, ****p , 0.0001.

Article Snippet: Commercially available Abs for detection of MAPK, a-tubulin, and b-actin were purchased as follows: AnxA5 (AF399; R&D Systems), AnxA13 (AF4149; R&D Systems), p-ERK (E10; Cell Signaling), ERK1 (MK12; BD), p-JNK (G9; Cell Signaling), JNK1/3 (C17; Santa Cruz Biotechnology), p-p38 (D3F9; Cell Signaling), p38a (5F11; Cell Signaling), a-tubulin (B-5-1-2; Sigma-Aldrich), and b-actin (AC-15; Abcam).

Techniques: Expressing, Incubation, Recombinant, Staining, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain.

doi: 10.4049/jimmunol.1401299

Figure Lengend Snippet: FIGURE 5. FPR family members are not involved in the recognition of AnxA5 and AnxA13 on BMDCs. (A) Immunoblot analysis of MAPK activation of BMDCs after incubation with 500 nM of the indicated recombinant protein for the indicated times. Lysates of CpG- and LPS-stimulated BMDCs from Tlr42/2 and WT mice, respectively, served as positive controls. Data are representative of three independent experiments. (B) GM-CSF–differentiated BMDCs from Tlr42/2 mice were incubated with the indicated concentrations of recombinant protein in the presence or absence of the FPR antagonist Boc-2 or apoptotic Jurkat T cells (aJ). Six to eight hours later, cells were stimulated with 40 nM CpG o/n. IL-12p40 concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of three independent experiments. *p , 0.05, ****p , 0.0001.

Article Snippet: Commercially available Abs for detection of MAPK, a-tubulin, and b-actin were purchased as follows: AnxA5 (AF399; R&D Systems), AnxA13 (AF4149; R&D Systems), p-ERK (E10; Cell Signaling), ERK1 (MK12; BD), p-JNK (G9; Cell Signaling), JNK1/3 (C17; Santa Cruz Biotechnology), p-p38 (D3F9; Cell Signaling), p38a (5F11; Cell Signaling), a-tubulin (B-5-1-2; Sigma-Aldrich), and b-actin (AC-15; Abcam).

Techniques: Western Blot, Activation Assay, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain.

doi: 10.4049/jimmunol.1401299

Figure Lengend Snippet: FIGURE 6. AnxA5 and AnxA13 suppress the induction of CD8+ T cell immune responses in vivo. (A) GM-CSF–differentiated BMDCs from WT mice were incubated with the indicated ratio of apoptotic Jurkat T cells (aJ) or apoptotic neutrophils (aNF, after 1 d in vitro culture). Four hours later, cells were stimulated with 1 ng/ml LPS o/n. TNF-a concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of three independent experiments. (B) aS2 mOVA, together with aS2 AnxA1, aS2 AnxA5, or aS2 AnxA13 were injected i.v. into mice. Mice injected with aS2 mock served as a negative control. After 8 d, the induction of OVA-specific CD8+ T cells in the mesenteric lymph nodes was analyzed by flow cytometry. Errors bars represent mean 6 SEM. (C and D) One day after transfer of 1 3 106 CFSE-labeled OT-I T cells, C57BL/6 WT mice were immunized i.v. with 0.5 3 106

Article Snippet: Commercially available Abs for detection of MAPK, a-tubulin, and b-actin were purchased as follows: AnxA5 (AF399; R&D Systems), AnxA13 (AF4149; R&D Systems), p-ERK (E10; Cell Signaling), ERK1 (MK12; BD), p-JNK (G9; Cell Signaling), JNK1/3 (C17; Santa Cruz Biotechnology), p-p38 (D3F9; Cell Signaling), p38a (5F11; Cell Signaling), a-tubulin (B-5-1-2; Sigma-Aldrich), and b-actin (AC-15; Abcam).

Techniques: In Vivo, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay, Injection, Negative Control, Cytometry, Labeling

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