Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the hybrid and β-I domains of wild-type and Alfa-tagged Integrin β1. b , Schematics showing the effect of 12G10 and Mab13 antibodies on integrin β1 activation state. These antibodies act by stabilizing the integrins in their active or inactive state. c , Quantification of integrin β1 uptake by flow cytometry using EndoNB in the presence of no antibodies (no treatment), or in the presence of 12G10 (activating), Mab13 (inactivating) or unrelated (anti integrin αvβ3 antibody). (n = 12, each n represents the median fluorescence of 2000-8000 cells). d , Structural alignment of the extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. See for comparison of Alfa-tagged dimeric and monomeric TFR. e , Schematics showing the experimental design to test if transferrin receptor uptake is stimulated by the presence of transferrin. f , Quantification of transferrin receptor uptake for 15 or 30 minutes by flow cytometry using EndoNB in the presence of increasing concentrations of transferrin. (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for quantification of transferrin-AlexaFluor488 signals at each condition. g , Structural alignment of the transmembrane and extracellular region of wild-type and Alfa-tagged AXL. Both structures generated with AlphaFold 3. h , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1ug/ml). (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for comparison with the conditions with and without 3C. ns = non-significant, * p> 0.05, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Generated, Comparison

Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the monomeric and dimeric extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR, PDB). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. b , Quantification of transferrin (AlexaFluor488) in each condition measured in . c , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1µg/ml) and also in the presence or not of 3C. (n = 4, each n represents the median fluorescence of 2000-8000 cells). ns = non-significant, ** p> 0.01, *** p> 0.001, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Generated, Flow Cytometry, Fluorescence

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Expressing, Recombinant, Produced, Transfection, Control, Activity Assay

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Construct, Expressing, Flow Cytometry, Staining, Concentration Assay, Control, Membrane, Phospho-proteomics, Western Blot

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Imaging, Construct, Inhibition, Membrane, Fluorescence

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Phospho-proteomics, Construct, Binding Assay, Mutagenesis, Comparison, Ligand Binding Assay, Activation Assay

Journal: Cancers

Article Title: Inhibition of AXL and VEGF-A Has Improved Therapeutic Efficacy in Uterine Serous Cancer

doi: 10.3390/cancers13235877

Figure Lengend Snippet: Mechanistic display of how inhibition of VEGF-A and AXL negatively regulate angiogenesis. This figure displays the mechanism between USC cells and HUVECs when AXL and VEGF-A are inhibited. AVB-500 blocks GAS6 from binding to AXL downregulating angiogenic factors. Simultaneously, both AVB-500 and bevacizumab inhibit activation of AXL and the VEGF receptor on the HUVECs.

Article Snippet: Western blots were probed with primary antibodies against AXL (1:1000, #8661) phospho-AXL Tyr702 (1:500, #5724), AKT (1:1000, #9272), and phospho-AKT Ser473 (1:500, #9271) from Cell Signaling Technology and GAS6 (1:1000, #AF885) from R&D Systems and corresponding secondary peroxidase-conjugated anti-rabbit IgG (H + L) (Jackson ImmunoResearch, # 711-035-152), anti-mouse IgG (H + L) (Jackson ImmunoResearch, # 711-035-150), or anti-goat IgG (H + L) (Jackson ImmunoResearch, # 711-035-180) ( ).

Techniques: Inhibition, Binding Assay, Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: AXL and GnRH receptor-like immunoreactivity in murine and human pituitaries. A Representative images showing AXL-like (red) and GnRH receptor-like (green) immunostaining in human, male C57 murine, and C57 female murine pituitary sections. Cell nuclei (blue) blue are shown in the merged images. B Frequency of overlapping AXL- and GnRH receptor-like immunoreactivity in a human pituitary; > 100 cells were analyzed from 7 different images for both AXL- and GnRH receptor-like immunoreactivity. C Representative images showing Gas6-like (red) and GnRH receptor-like (green) immunostaining in human, male C57 murine, and C57 female murine pituitary sections. Cell nuclei (blue) blue are shown in the merged images. D Representative images showing AXL-like (red) and Gas6-like (green) immunostaining in a human pituitary section. Cell nuclei (blue) blue are shown in the merged images. Images are representative of ≥ 3 paraffin sections; scale bars = 20 μm

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 µg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 µg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Immunostaining

Journal: Cell Communication and Signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: Gas6-dependent AXL receptor activation enhances GnRH receptor signaling processes. A Proportion of phosphorylated ERK relative to total ERK (pERK/ERK) in αT3-1 and LβT2 cells under control conditions and after incubation with Gas6 (100 nM for 3 h); n = 3 for each condition. B Time course of Gas6-dependent changes (100 nM) in pERK/ERK in LβT2 cells (from left ); effect of incubating LβT2 cells with GnRH (10 nM) and GnRH plus Gas6 (100 nM) for 5 min on pERK/ERK (at right ); n = 3 for each condition. C Time course of Gas6-dependent (100 nM) changes on Egr-1 transcript abundance in αT3-1 and LβT2 cells; n = 3. D LHβ protein abundance in the culture media and lysates of LβT2 cells under control conditions and after 2 h incubations with Gas6 (100 nM) and GnRH (10 nM); n = 3. € LHβ protein abundance in LβT2 culture media under control conditions and after 2 h incubations with Gas6 (100 nM), GnRH (10 nM), the GnRH receptor antagonist cetrorelix (2 nM), the AXL receptor antagonist R428 (50 µM), and the MEK inhibitor U0126 (10 µM); n = 3 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; * P < 0.05

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 µg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 µg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Activation Assay, Control, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: AXL and GnRH receptor stimulation promotes pro-MMP9 release. Pro-MMP9 abundance in the culture media and lysates of αT3-1 ( A ) and LβT2 ( B ) cells following incubation with Gas6 (100 nM), GnRH (10 nM), Gas6 plus GnRH, and the AXL receptor antagonist R428 (50 µM), the MEK inhibitor U0126 (10 µM). n = 4 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; ns = P > 0.05; * P < 0.05

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 µg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 µg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: AXL and GnRH receptor activation promotes migration of clonal gonadotropes. Transwell migratory responses (% cells) of αT3-1 ( A ) and LβT2 ( B ) cells following 48 h incubations with Gas6 (100 nM), GnRH (10 nM), Gas6 plus GnRH, the MEK inhibitor U0126 (10 µM), and the MEK activator PAF C-16 (100 µM). n = 4 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; ns = P > 0.05; * P < 0.05. C Working model of AXL and GnRH receptor signaling integration in gonadotropes

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 µg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 µg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Activation Assay, Migration

Journal: HemaSphere

Article Title: AXL Inhibition Extinguishes Primitive JAK2 Mutated Myeloproliferative Neoplasm Progenitor Cells

doi: 10.1097/HS9.0000000000000233

Figure Lengend Snippet: GAS6 levels in MPN ; A, Levels of GAS6 were measured in plasma of normal and JAK2 V617F patients by ELISA and the results expressed as ng/ml of plasma (mean ± SEM, n = 6). RNA expression levels in CD34 + cells were measured using Illumina HiSeq NGS and data shown as the number of normalized RNA reads observed. Results for housekeeping genes (B) and GAS6 (C) are shown (mean ± SEM, n = 3 normals, n = 7 for JAK2 V617F). D, CD34 + cells isolated from MPN patients and healthy controls were incubated with 20 μM DCFDA to assess ROS levels. Amalgamated flow cytometry data is displayed as median fluorescent intensity ± SEM (n = 3 normals, n = 7 for JAK2 V617F). The results of a t test are shown, ns indicates not significant.

Article Snippet: Levels of GAS6 were measured using the Human GAS6 Duoset ELISA from R&D Systems (DY885B) as per manufactures instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, RNA Expression, Isolation, Incubation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization

doi: 10.1172/JCI125505

Figure Lengend Snippet: (A) Gas6 levels in the BAL fluid from an RSV-infected lung of a WT mouse. (B) Immunohistochemical analysis (DAB) of Gas6 in the tissue sections from a naive (D0) and RSV-infected lung. Blue arrows indicate Gas6+ macrophages and green arrows indicate Gas6+ epithelial cells. Scale bar: 200 μm. (C) Gas6 levels in the culture supernatant of alveolar macrophages isolated from naive and RSV-infected mice on days 4, 8, and 12 after infection. (D) WT and Gas6–/– mice were infected with S. pneumoniae at day 8 after RSV infection and euthanized on days 1, 2, 3, and 6 after S pneumoniae infection. (E) Titers of RSV in the whole lung from RSV-infected mice on days 4 and 8 after RSV infection. (F) Changes in the survival rate and (G) body weight of RSV-infected WT or Gas6–/– mice after S. pneumoniae infection. (H) Titers of S. pneumoniae in the BAL fluid from RSV-infected mice on days 1 and 3 after S. pneumoniae infection. (I) Representative H&E-stained lung tissue sections on day 1 after S. pneumoniae infection. Scale bars: 200 μm (upper), 50 μm (lower). (J) Number of neutrophils, macrophages, and lymphocytes in the BAL fluid on day 1 after S. pneumoniae infection. (K) The levels of IFN-γ, NO, TNF-α, IL-1β, CXCL1, and CXCL2 in the BAL fluid from WT and Gas6–/– mice on day 1 after S. pneumoniae infection. The data are expressed as mean ± SEM; n = 10 (F and G), n = 4–6 (except for F and G). Representative results from 2 independent experiments are shown. The following statistical tests were used: 1-way ANOVA (A, C, H, J, and K), 2-tailed Student’s t test (E), and Gehan-Breslow-Wilcoxon test (F). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The levels of human Gas6 and IFN-γ in patient supernatants were determined using the Duoset ELISA development kit (R&D Systems), following the manufacturer’s instructions.

Techniques: Infection, Immunohistochemical staining, Isolation, Staining

Journal: The Journal of Clinical Investigation

Article Title: Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization

doi: 10.1172/JCI125505

Figure Lengend Snippet: (A) Mice were infected with RSV or treated with recombinant Gas6 (rGas6; 1 μg/dose, 3 doses) before S. pneumoniae infection on day 8 after RSV infection. Animals were euthanized for analysis on days 1, 2, and 3 after S. pneumoniae infection. (B) Changes in the survival rate and (C) body weight after S. pneumoniae infection. (D) Titers of S. pneumoniae in the BAL fluid from RSV-infected or rGas6-injected mice on days 1 and 3 after S. pneumoniae infection. (E) Representative H&E-stained lung tissue sections on day 1 after S. pneumoniae infection. Scale bars: 200 μm (upper), 50 μm (lower). (F) Numbers of inflammatory cells in the BAL fluid from both treatment groups on day 1 after S. pneumoniae infection. (G) The levels of IFN-γ, NO, TNF-α, IL-1β, CXCL1, and CXCL2 in the BAL fluid from both treatment groups on day 1 after S. pneumoniae infection. The data are expressed as the mean ± SEM; n = 10 (B and C), n = 4–6 (except for B and C). Representative results from 2 independent experiments are shown. The following statistical tests were used: Gehan-Breslow-Wilcoxon test (B), 1-way ANOVA (C, D, F, and G). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The levels of human Gas6 and IFN-γ in patient supernatants were determined using the Duoset ELISA development kit (R&D Systems), following the manufacturer’s instructions.

Techniques: Infection, Recombinant, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization

doi: 10.1172/JCI125505

Figure Lengend Snippet: (A) The levels of IL-18 in the BAL fluid on day 1 after S. pneumoniae infection after RSV infection in various treatment groups (WT vs. Gas6–/–; WT vs. Axl–/–; IgG vs. Axl mAb treatment; hydroxypropyl methylcellulose vs. BGB324 treatment; PBS vs. rGas6 treatment; and control liposome vs. clodronate liposome treatment). The levels of IFN-γ, NO, and TNF-α in the BAL fluid from each group at day 1 after S. pneumoniae infection in the anti–IL-18 mAb-treated (B) and recombinant IL-18–treated (C) mice infected with S. pneumoniae and/or RSV. (D and E) IL-18 production in the alveolar macrophages isolated from WT and Gas6–/– mice on day 8 after RSV infection and stimulated with S. pneumoniae for 1 hour. (F) Activation of caspase-1 and degradation of IκB in the alveolar macrophages isolated from WT and Gas6–/– mice on day 8 after RSV infection and stimulated with S. pneumoniae for 3 hours (left panel). The ratios of cleaved caspase-1 (p10) and the IκB to GAPDH ratio are shown (right panel). The data are expressed as mean ± SEM; n = 4–6 (A and B), n = 2–3 (E and F). Representative results from 2 independent experiments are shown. The following statistical tests were used: 2-tailed Student’s t-test (A, for WT vs. Gas6–/–; WT vs. Axl–/–; IgG vs. Axl mAb treatment; hydroxypropyl methylcellulose vs. BGB324 treatment), and 1-way ANOVA (A, for PBS vs. rGas6 treatment; and control liposome vs. clodronate liposome treatment, B, C, and E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The levels of human Gas6 and IFN-γ in patient supernatants were determined using the Duoset ELISA development kit (R&D Systems), following the manufacturer’s instructions.

Techniques: Infection, Recombinant, Isolation, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization

doi: 10.1172/JCI125505

Figure Lengend Snippet: Shortly after RSV infection, the epithelial cells and alveolar macrophages produce Gas6. The Gas6/Axl axis polarizes antibacterial (M0-like) macrophages into nonantibacterial (M2-like) macrophages that inhibit the activation of caspase-1/IL-18. The low levels of IL-18/IFN-γ in the macrophages and NK cells result in severe secondary pneumococcal infection.

Article Snippet: The levels of human Gas6 and IFN-γ in patient supernatants were determined using the Duoset ELISA development kit (R&D Systems), following the manufacturer’s instructions.

Techniques: Infection, Activation Assay