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: Scientific Reports

Article Title: Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

doi: 10.1038/srep36520

Figure Lengend Snippet: ( A ) mRNA expression in control and Gas6 overexpressed PCa cells. mRNA was extracted 2 days after serum starvation. *p < 0.05, **p < 0.01 compared to PC3 Control cells. ## p < 0.01 compared to DU145 Control cells. ( B ) mRNA expression in sh control and sh Gas6 knockdown PCa cells. mRNA was extracted after 6 days of culture in 0.5% FBS. Culture media was replaced at day3.*p < 0.05, **p < 0.01 compared to PC3 sh Control cells. ## p < 0.01 compared to DU145 sh Control cells. ( C–E ) mRNA expression in PCa cells 6 days after siRNA treatments targeting Axl. *p < 0.05, **p < 0.01 compared to si scramble control PC3cells, ## p < 0.01 compared to si scramble control DU145 cells. ( F,G ) Western blots showing protein expression levels of Axl, TGFBR2 and TGFBBR3 in PCa cells after the reduction of Axl or Gas6 by shRNA ( F ) or siRNA ( G ). Protein samples were loaded 6 days after siRNA treatments targeting Axl (G) .

Article Snippet: An antibody sandwich ELISA was used to evaluate Gas6 expression in the conditioned media and cell lysates by the following the directions of manufacturer (Cat. #DY986 and DY885, R&D Systems, Minneapolis, MN).

Techniques: Expressing, Control, Knockdown, Western Blot, shRNA

Journal: Scientific Reports

Article Title: Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

doi: 10.1038/srep36520

Figure Lengend Snippet: ( A ) Relative mRNA levels in PCa cells were evaluated 24 hours after TGF-β1 (Cat #: 240-B/CF, R&D Systems, Minneapolis, MN) or TGF-β2 (Cat #: 302-B2/CF, R&D Systems, Minneapolis, MN) stimulation (both 2 ng/ml). *p < 0.05, **p < 0.01 compared to PCa cells without TGF-β stimulation, # p < 0.05, ## p < 0.01 compared to PCa with TGF-β1 stimulation. ( B,C ) PCa cells were pre-labeled with DiD, and DiD retention was evaluated 6 days after TGF-β2 stimulation by flow cytometry. *p < 0.05, **p < 0.01 compared to PC3 cells without TGF-β2 treatments, ## p < 0.01 compared to DU145 cells without TGF-β2 treatments in each of sh Control, sh Axl or sh Gas6 cells. ( D ) Western blots showing p27 expression levels in PCa cells 3 days after TGF-β2 treatment. ( E ) Relative p27 expression levels evaluated by densitometry. P27 expression was normalized by β-actin expression. *p < 0.05, **p < 0.01 compared to PCa sh Control cells without TGF-β2 treatments. # p < 0.05, ## p < 0.01 compared to DU145 sh Axl cells without TGF-β2 stimulation.

Article Snippet: An antibody sandwich ELISA was used to evaluate Gas6 expression in the conditioned media and cell lysates by the following the directions of manufacturer (Cat. #DY986 and DY885, R&D Systems, Minneapolis, MN).

Techniques: Labeling, Flow Cytometry, Control, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

doi: 10.1038/srep36520

Figure Lengend Snippet: Gas6 produced by osteoblasts binds to Axl expressed by disseminated PCa cells, and its signaling induces expression of both TGF-β ligands (TGF-β1 and TGF-β2) and their receptors (TGFBR2 and TGFBR3). Subsequently, autocrine and paracrine TGF-β signaling induces PCa dormancy.

Article Snippet: An antibody sandwich ELISA was used to evaluate Gas6 expression in the conditioned media and cell lysates by the following the directions of manufacturer (Cat. #DY986 and DY885, R&D Systems, Minneapolis, MN).

Techniques: Produced, Expressing