Journal: PLoS ONE

Article Title: Neuroinductive properties of mGDNF depend on the producer, E . Coli or human cells

doi: 10.1371/journal.pone.0258289

Figure Lengend Snippet: (a), (b), (c) and (d) Analysis of the isolated factors using the rat model of whole embryonic spinal ganglion culture supplemented with: (a) cmGDNF (mGDNF concentrated from the medium conditioned by transgenic cells); (b) no factors (control); (c) recombinant mGDNF/Coli; (d) recombinant Pro-mGDNF/Coli. Scale bar 500 μm. (e) Quantitation of ganglion cell processes. cmGDNF was isolated by immunoprecipitation from the medium conditioned by HEK293 cells; mGDNF, recombinant mGDNF/Coli; Pro-mGDNF, recombinant Pro-mGDNF/Coli; recGDNF, positive control; control, negative control with no factors. The results are presented as the mean ±SD (*p<0,05 vs control) (N = 3).

Article Snippet: The mGDNF protein was isolated from the medium conditioned by HEK293 cells expressing mGDNF/GFP by immunoprecipitation using GFP-Trap agarose (Chromotek, Germany).

Techniques: Isolation, Transgenic Assay, Control, Recombinant, Quantitation Assay, Immunoprecipitation, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: Neuroinductive properties of mGDNF depend on the producer, E . Coli or human cells

doi: 10.1371/journal.pone.0258289

Figure Lengend Snippet: HEK/GDNF, HEK293 cells transfected with mGDNF; cmGDNF, mGDNF concentrated from medium containing mGDNF; GDNF1-Coli, mGDNF isolated from E . coli by the standard method; GDNF2-Coli, mGDNF isolated from E . coli with refolding using the mouse serum.

Article Snippet: The mGDNF protein was isolated from the medium conditioned by HEK293 cells expressing mGDNF/GFP by immunoprecipitation using GFP-Trap agarose (Chromotek, Germany).

Techniques: Transfection, Isolation

Journal: PLoS ONE

Article Title: Neuroinductive properties of mGDNF depend on the producer, E . Coli or human cells

doi: 10.1371/journal.pone.0258289

Figure Lengend Snippet: (a) The number of tyrosine hydroxylase-immunopositive neurons in the ventral midbrain in intact animals and those pre-administered with the GDNF variants into the striatum 20 h prior to MPTP exposure for 14 days. (b) Motor coordination of experimental animals administered with recombinant mGDNF and control saline solution with the subsequent MPTP injection. The Contr and GDNF1-Coli values significantly differ (p≤0,05) from those of cmGDNF and и GDNF2-Coli. intact, no exposure; MPTP, exposure to MPTP alone; Contr, control saline solution without GDNF; НЕК/GDNF, HEK293 cells transfected with mGDNF; НЕК/GFР medium conditioned by GFP-transgenic cells; cmGDNF, mGDNF concentrated from the conditioned medium; GDNF1-Coli, mGDNF isolated from E . coli by the standard method; GDNF2-Coli, mGDNF isolated from E . coli with refolding using the mouse serum. The results are presented as the mean ±SD (*p<0,05 vs control) (N = 5–7 per group).

Article Snippet: The mGDNF protein was isolated from the medium conditioned by HEK293 cells expressing mGDNF/GFP by immunoprecipitation using GFP-Trap agarose (Chromotek, Germany).

Techniques: Recombinant, Control, Saline, Injection, Transfection, Transgenic Assay, Isolation

Journal: PLoS ONE

Article Title: Neuroinductive properties of mGDNF depend on the producer, E . Coli or human cells

doi: 10.1371/journal.pone.0258289

Figure Lengend Snippet: The analysis of biological effects of GDNF produced by bacterial expression system ( E . coli) and mammalian expression system (HEK293) [ 20 , 21 ].

Article Snippet: The mGDNF protein was isolated from the medium conditioned by HEK293 cells expressing mGDNF/GFP by immunoprecipitation using GFP-Trap agarose (Chromotek, Germany).

Techniques: Produced, Expressing, Modification, In Vivo, Recombinant, Negative Control, Positive Control

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 3 Effect of grape skin fermentation solution on cell growth. (A) Effect of treatment with grape skin fermentation solution for 48 hours on 293T cell growth and viability. (B) Effect of treatment with grape skin fermentation solution for 48 hours on HepG2 cell growth and viability (CF: the fermentation solution fermented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques:

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 4 Effect of treatment grape skin fermentation solution for 48 hours on 293T cell morphology (200) (CF: the fermentation solution fer- mented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques:

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 6 SOD, GSH, CAT, and GSH-Px mRNA expressions in 293T cells. a–eMean values with different letters in the same column differed significantly (P < 0.05) by Duncan's multiple range test. Values presented are the means standard deviation (N ¼ 3/group) (CF: the fermentation solution fermented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques: Standard Deviation

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 7 SOD, GSH, CAT, and GSH-Px protein expression in 293T cells. Values presented are the means standard deviation (N ¼ 3/group) (CF: the fermentation solution fermented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol) (* stands P < 0.05, ** stands 0.05 < P < 0.01, **** stands 0.01 < P < 0.001, **** stands 0.001 < P < 0.0001).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: A Viral-Encoded Homologue of IPS-1 Modulates Innate Immune Signaling During KSHV Lytic Replication

doi: 10.1101/2024.07.24.604690

Figure Lengend Snippet: (A) To determine if v-IPS-1 was indeed a protein produced during KSHV infection, as well as determine where in the latent and lytic parts of the viral replication cycle we developed an antibody was against the unique C terminus of v-IPS-1 as indicated. This amino acid sequences had no similarity to any human or any other KSHV protein. (B) An expression clone of the v-IPS-1 ORF that was PCR amplified from a cDNA library from KSHV infected BCBL1 cells was transfected into HEK 293T cells and whole cells protein lysates were analyzed by western blot using the the anti-v-IPS-1 antibody developed in 2A as well as an anti-Actin antibody. Only cells transfected with the v-IPS-1 expression plasmid showed a band reactive to the v-IPS-1 antibody. To the right of blot is a graph of quantitation of 8 replicates of this experiment with **** standing for p of <0.0001 by unpaired t-test. (C) BCBL-1 cells which hold the latent KSHV genome and were engineered to express RTA under control of a tetracycline inducible promoter (TREx) were analyzed by flow cytometry for the lytic marker ORF59 and the antibody to v-IPS-1 from 2A. BJAB cells, being used as a negative control without KSHV, and BCBL-1 cells with a TREx expressing a vector control as a cell that should not enter lytic phase were also used. Only ORF59+ BCBL-1 cells which are actively in lytic replication express v-IPS-1. (D) Cells in 2C were also stained for ORF59 and the histogram marked with * confirms that high ORF59 expression is in cells expressing v-IPS-1. (E) Cells in 2C were also stained for LANA expression. We confirmed that our BCBL-1 cells do have KSHV latently infected. (F) Three biologic replicates of the flow cytometry shown in 2C were quantitated and the percentage of v-IPS-1 positive cells were graphed. This again confirms that only lytic replicating KSHV infected cells express v-IPS-1.

Article Snippet: Human embryonic kidney 293T cells (ATCC Catalogue number were grown in Dulbeco’s Modified Eagle Medium (DMEM, high glucose) from Thermo Fisher.

Techniques: Produced, Infection, Expressing, Amplification, cDNA Library Assay, Transfection, Western Blot, Plasmid Preparation, Quantitation Assay, Control, Flow Cytometry, Marker, Negative Control, Staining

Journal: bioRxiv

Article Title: A Viral-Encoded Homologue of IPS-1 Modulates Innate Immune Signaling During KSHV Lytic Replication

doi: 10.1101/2024.07.24.604690

Figure Lengend Snippet: (A) A diagram representing the interferon induction pathways showing convergence at TRAF3. To determine how v-IPS-1 impacted the different steps of the IFN-beta induction signaling pathway we overexpressed v-IPS-1 along with cellular components of the pathway. (B) HEK 293T cells were transfected with an expression plasmid for IPS-1, which will induce the co-transfected IFN-beta luciferase plasmid which has luciferase under control of the IFN-beta promoter. In addition, these cells were co-transfected with or without the v-IPS-1 expression plasmid as well as a Renilla luciferase reporter. After 18 hours, cells were lysed and firefly and Renilla luciferase was quantitated, with average of triplicates graphed with standard deviations reflected in the error bars. Here v-IPS-1 blocks induction of IFN-beta by IPS-1 overexpression. As in (B), expression plasmids for constitutively active RIG-I and MDA-5 were used in (C), TBK1 in (D) and IRF3 or IRF7 in (E) to see the impact of v-IPS-1 on these different inducers of IFN-beta. Expression of v-IPS-1 blocks interferon induction upstream of TRAF3 when stimulated with either RIG-I, MDA5 or IPS-1. However, v-IPS-1 does not block IFN-beta induction when stimulated by overexpression of TBK1, IRF3 or IRF7, all of which are downstream of TRAF3. Downward arrows are used to mark low values on graphs to point them out in the graphs. All data is representative of technical triplicates and was repeated at least three times.

Article Snippet: Human embryonic kidney 293T cells (ATCC Catalogue number were grown in Dulbeco’s Modified Eagle Medium (DMEM, high glucose) from Thermo Fisher.

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Over Expression, Blocking Assay

Journal: bioRxiv

Article Title: A Viral-Encoded Homologue of IPS-1 Modulates Innate Immune Signaling During KSHV Lytic Replication

doi: 10.1101/2024.07.24.604690

Figure Lengend Snippet: To determine how v-IPS-1 impacts the stability of signaling nodes in the IFN induction pathway, we expressed v-IPS-1 along with different cellular proteins. (A) HEK 293T cells were transfected with an expression plasmid for FLAG-IPS-1 with or without the v-IPS-1 expression plasmid and after 24 hours, cells were lysed for Western blot of FLAG-IPS-1 and Actin. The smaller 52 kDa form of IPS-1 can also be seen in lane 2. Below the blot is a graph of quantitation of 5 replicates of this experiment with *** standing for p of 0.0007 by unpaired t-test. As in (B), expression plasmids HA-TRAF3 were transfected along with or without a v-IPS-1 expression plasmid. After 24 hours, cells were lysed for Western blot of HA-TRAF3 and Tubulin. Below the blot is a graph of quantitation of 5 replicates of this experiment with *** standing for p of 0.0003 by unpaired t-test. (C) Since IPS-1 and TRAF3 often interact, we sought to determine if TRAF3 could overcome v-IPS-1 destabilization of IPS-1. Combinations of HA-TRAF3, FLAG-IPS-1 and v-IPS-1 were transfected into HEK 293T cells and after 24 hours, cells were lysed for Western blotted for FLAG-IPS-1 and Actin. (D) To determine the importance of membrane targeting of IPS-1 on v-IPS-1 destabilization of IPS-1, we used a construct of FLAG-IPS-1 that was missing the transmembrane domain. HEK 293T cells were transfected with FLAG-IPS-1 ΔTM with or without the v-IPS-1 expression plasmid and after 24 hours, cells were lysed for Western blotted for FLAG-IPS-1 ΔTM and Tubulin. To the right of blot is a graph of quantitation of 5 replicates of this experiment with * standing for p of 0.01 by unpaired t-test. (E and F) Similar to the above experiments, Full length FLAG-RIG-I (E) and HA-TBK1 (F) were co-transfected with v-IPS-1 and after 24 hours cells were lysed for Western blotted for FLAG-RIG-I (E) or HA-TBK1 (F) and Tubulin. Graphs of normalized bands whose intensities were quantitated in 3 replicates of this experiment were plotted with ns in (E) standing for no significance and * in (F) standing for p of 0.04 by unpaired t-test.

Article Snippet: Human embryonic kidney 293T cells (ATCC Catalogue number were grown in Dulbeco’s Modified Eagle Medium (DMEM, high glucose) from Thermo Fisher.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitation Assay, Membrane, Construct

Journal: bioRxiv

Article Title: A Viral-Encoded Homologue of IPS-1 Modulates Innate Immune Signaling During KSHV Lytic Replication

doi: 10.1101/2024.07.24.604690

Figure Lengend Snippet: To determine how v-IPS-1 impacts the TRIF signaling, we expressed v-IPS-1 along with components of TRIF signaling. (A) HEK 293T cells were transfected with an expression plasmid for Myc-tagged with or without the v-IPS-1 expression plasmid and after 24 hours, cells were lysed Western blotted for Myc-TRIF and Actin was done. To the right of blot is a graph of quantitation of 5 replicates of this experiment with ** standing for p of 0.0075 by unpaired t-test. (B) Similar to , HEK 293T cells were transfected with an expression plasmid for Myc-tagged TRIF with or without the v-IPS-1 expression plasmid and after 24 hours, cells were lysed and firefly and Renilla luciferase was quantitated, with average of triplicates graphed. Refer to for diagram of location of TRIF in signaling pathways. Downward arrows are used to mark low values on graphs. (C) HEK 293T cells were transfected with an expression plasmid for HA-tagged TRAF6 with or without the v-IPS-1 expression plasmid and after 24 hours, cells were lysed Western blotted for HA-TRAF6 and Tubulin. The Western blots is representative and part of a set of 3 biological replicates.

Article Snippet: Human embryonic kidney 293T cells (ATCC Catalogue number were grown in Dulbeco’s Modified Eagle Medium (DMEM, high glucose) from Thermo Fisher.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitation Assay, Luciferase, Protein-Protein interactions

Journal: bioRxiv

Article Title: A Viral-Encoded Homologue of IPS-1 Modulates Innate Immune Signaling During KSHV Lytic Replication

doi: 10.1101/2024.07.24.604690

Figure Lengend Snippet: To determine if v-IPS-1 could impact establishment of an antiviral state in cells, we used infection with VSV that was engineered to express GFP. IFN expression blocks VSV replication and in this case should limit GFP expression. HEK 293T cells in A, B and C were infected with VSV-GFP. While cells in (A) were not transfected prior to infection, cells in (B) were transfected with IPS-1 to induce an antiviral state and cells in (C) were transfected with the v-IPS-1 expression plasmids to assess the ability of v-IPS-1 to block antiviral activity during VSV infection. Images of cells were taken, and representative images are shown. The bar represents 400 mm. GFP expression indicated levels of virus replication. (D) Cells from A, B and C were fixed and analyzed by flow cytometry to monitor the percentage of infected cells which is indicated by GFP produced by VSV replication. Downward arrows are used to mark low values on graphs. (E) To determine if v-IPS-1 blocks signaling from external or exogenous IFN, we stimulated v-IPS-1 expressing cells with IFN. HEK 293T cells were transfected with or an empty expression plasmid or one expressing v-IPS-1 expression plasmid cells as well as a firefly luciferase reporter under the control of the Interferon-stimulated response element (ISRE) promoter and Renilla luciferase reporters for 18 hours. ISRE promoters are induced by exogenous IFN stimulation. After an additional 8 hours of stimulation by Interferon-alpha, cells were lysed and firefly and Renilla luciferase was quantitated, with average of triplicates graphed.

Article Snippet: Human embryonic kidney 293T cells (ATCC Catalogue number were grown in Dulbeco’s Modified Eagle Medium (DMEM, high glucose) from Thermo Fisher.

Techniques: Infection, Expressing, Transfection, Blocking Assay, Activity Assay, Virus, Flow Cytometry, Produced, Plasmid Preparation, Luciferase, Control

Journal: Molecular Cancer

Article Title: Transient intracellular expression of PD-L1 and VEGFR2 bispecific nanobody in cancer cells inspires long-term T cell activation and infiltration to combat tumor and inhibit cancer metastasis

doi: 10.1186/s12943-025-02253-6

Figure Lengend Snippet: Efficacy of FAP1V2 transient expression in mediating PD-1 checkpoint blockade immune inhibition of LLC progress in mice models. A Schematic diagram and ( B ) flow cytometry assay showed that the blockage of cell surficial PD-L1 by FAP1V2 or FAP1 caused reduced activity of PD-L1 to bind commercial PE-labeled human PD-1. The cells were transfected with pEGFP-C1 , pEGFP-C1-AP1 or pEGFP-C1-AP1V2 , respectively. The cells were not treated with 0.1% Triton before cell cytometry analysis. C to ( F ) Cell flow cytometry and antibody competition assays demonstrated that intracellular antibodies FAP1 and FAP1V2, or FAV2 and FAP1V2 could selectively bind to PD-L1 or VEGFR2 expressed by LLC cells. Initially, 293 T cells were separately transfected with pEGFP-C1-AP1 , pEGFP-AV2 or pEGFP-C1-AP1V2 . Following co-incubation of their lysates with LLC cells (fixed with paraformaldehyde), competitive binding assays were performed using commercial anti-mouse PD-L1 and VEGFR2 antibodies (conjugated with APC) against the corresponding antigens. Panel (C) shows LLC cells in four conditions, from left to right: Control: No co-incubation with 293 T lysates or commercial antibody. Anti-PD-L1 antibody: Co-incubated with commercial anti-mouse PD-L1 antibody. Single-target intrabody (FAP1) plus anti-PD-L1 antibody: Co-incubated with 293 T lysate expressing single-target intrabody FAP1, followed by addition of commercial anti-mouse PD-L1 antibody. Double-target intrabody (FAP1V2) plus anti-PD-L1 antibody: Co-incubated with 293 T lysate expressing double-target intrabody FAP1V2, followed by addition of commercial anti-mouse PD-L1 antibody. D Column graph illustrates the average fluorescence values of results shown in ( C ). Panel ( E ) shows LLC cells in four conditions, from left to right: Control: No co-incubation with 293 T lysates or commercial antibody. Anti-VEGFR2 antibody: Co-incubated with commercial anti-mouse VEGFR2 antibody. Single-target intrabody (FAV2) plus anti-PD-L1 antibody: Co-incubated with 293 T lysate expressing single-target intrabody FAV2, followed by addition of commercial anti-mouse VEGFR2 antibody. Double-target intrabody (FAP1V2) plus anti-VEGFR2 antibody: Co-incubated with 293 T lysate expressing double-target intrabody FAP1V2, followed by addition of commercial anti-mouse VEGFR2 antibody. F Column graph presents the average fluorescence values of results shown in ( E ). This experiment was independently repeated three times. Based on three mean fluorescence values (MFV) of the cell populations from each sample, as reported by flow cytometer (Channel APC-A), the data for the mean fluorescence values detected at Channel APC-A are presented as Mean MFV ± SEM. *, **, *** represent significant differences compared to the control group at the levels of P < 0.05, P < 0.01, P < 0.001, respectively. #, ##, ### indicate significant differences between treatment groups at the levels of P < 0.05, P < 0.01, P < 0.001, respectively. G to ( I ) The effect of anti-PD-L1-anti-VEGFR2 chimeric intrabody FAP1V2 on enhancing immune activity of suppressing tumorigenesis and growth of LLC cells in C57BL/6 mice. G Timeline for studying transient FAP1V2 expression mediated anticancer therapy via immune activation and antimetastatic effects. H Tumor size varied in the mice injected with LLC cancer cells which transiently expressed FAP1, FAV2 or FAP1V2 intrabody, respectively. The group of mice inoculated with LLC cells transiently transfected with blank plasmid ( pEGFP-C1 ) were set as a control. The LLC cells were respectively subcutaneously injected to the right armpits first and then to the left armpits 4 days later, which can maintain the transient expression of the intrabodies for total 6–8 days in the beginning. The data on tumor sizes for each group, which included tumors located in the same side of armpits from 6 mice, are presented as the Mean Tumor size ± SEM. (I) Images of the tumors separated from the right and left armpits of the mice which were sacrificed at Day 28

Article Snippet: The cells were incubated with 293T cell lysate containing FAP1, FAV2 or FAP1V2 at room temperature for 30 min, followed by incubation with commercial rabbit-derived Anti-mouse PD-L1-APC or Anti-mouse VEGFR2-APC (Elabscience, Wuhan, China) at room temperature for 30 min. After centrifugation at 2,000 × g for 4 min and removal of the supernatant, 200 μL of fresh medium was added for flow cytometry analysis (using APC detection channel).

Techniques: Expressing, Inhibition, Flow Cytometry, Activity Assay, Labeling, Transfection, Cytometry, Incubation, Binding Assay, Control, Fluorescence, Activation Assay, Injection, Plasmid Preparation

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: (A) WNT7A binds to RECK and FZD8. HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with indicated purified proteins (25 nM) for 72 hr and CM subjected to Protein A pull-down. (B) WNT3A binds to FZD4 and FZD8, but not to GPR124 and RECK. HEK293 RECK −/− WNT3A cells were cultured in medium supplemented with indicated purified proteins (25 nM) for 72 hr and CM subjected to Protein A pull-down. (C) Super TOP-Flash (STF) canonical Wnt/β-catenin reporter gene assay. HEK293 RECK +/+ or RECK −/− cells were co-transfected with the indicated expression constructs and STF/RLuc reporters. 24 hr after transfection sRECK-Fc protein was added at 0, 10, 100, or 1,000 nM (wedges). 24 hr later STF activity was normalized with RLuc activity. Mean (n = 3) ± SD. *p < 0.05 versus no protein. (D) WNT7A binding to RECK is mediated by RECK cystine knot motif (CK) and cysteine-rich domains (CRD). HEK293 RECK −/− WNT7A cell culture medium was supplemented with the indicated proteins (3 nM) and conditioned for 96 hr. CM were subjected to Protein A pull-down. (E and F) Cell surface RECK binds to WNT7A, but not to WNT3A. HEK293 RECK −/− WNT7A (E) or WNT3A (F) cells were transfected with the indicated expression constructs. 48 hr after transfection, SNAP tags were specifically biotinylated (bSNAP) and cell surface proteins cross-linked in situ using DTSSP (100 μM). Cells were lysed, subjected to streptavidin pull-down, and cross-links reversed. WCL, whole cell lysate. All western blots (WB) were performed under reducing conditions. All data are representative of at least two independent experiments with similar results. CM, conditioned medium; s, soluble ECD; Fc, IgG Fc fragment. See also and .

Article Snippet: Anti-mouse GPR124 ECD antiserum was generated by immunizing rabbits (Rockland Immunochemicals, Pottstown, PA) with recombinant mouse GPR124 ECD, which had been cloned, expressed, and purified from stably transfected HEK293 cell conditioned medium as previously described ( ).

Techniques: Cell Culture, Purification, Reporter Gene Assay, Transfection, Expressing, Construct, Activity Assay, Binding Assay, In Situ, Western Blot

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: (A) RECK and FZD8 only bind newly secreted WNT7A. Culture media of HEK293 RECK −/− WNT7A cells were supplemented with the indicated proteins (100 nM) and conditioned for 72 hr (“during conditioning”). Alternatively, the indicated proteins were added to the CM after harvesting after 72h (“after conditioning”). CM were subjected to Protein A pull-down. (B and C) CM from HEK293 RECK −/− WNT7A (B) or WNT3A (C) cells were analyzed by reducing (+DTT) or non-reducing (−DTT) SDS-PAGE/WB. Where indicated CM were subjected to Triton X-114 phase separation. (D) sRECK-Fc protein stabilizes newly secreted, monomeric, hydrophobic WNT7A. Culture medium of HEK293 RECK −/− WNT7A cells was supplemented with indicated proteins (100 nM) and conditioned for 72 hr (“during conditioning”). Alternatively, indicated proteins were added to CM after harvesting after 72 hr (“after conditioning”). CM aliquots were subjected to Triton X-114 phase separation and remaining CM to Protein A pull-down followed by Triton X-114 phase separation of pull-downs and unbound supernatants. Irrelevant lanes (between lanes 4 and 5) have been removed from the WB. (E) RECK and WNT7A form a 1:1 complex. Expi293F cells were co-transfected with sRECK-His and WNT7A. 96 hr after transfection CM was harvested and sRECK-His:WNT7A complex purified by combined chemical cross-linking (BS 3 )/tandem affinity purification. Purified complex was analyzed by reducing SDS-PAGE/WB. (F) Free, hydrophobic WNT7A is highly unstable. sRECK-Fc:WNT7A, sFZD8-Fc-Biotin:WNT7A, and sFZD8-Fc-Biotin:WNT3A complexes were isolated from the corresponding HEK293 RECK −/− WNT7A/WNT3A CM (96 hr, 100 nM recombinant protein) using Protein A or streptavidin agarose. Bound Wnt proteins were eluted by low pH, neutralized, diluted into PBS/10% FBS and incubated at 37°C. At indicated time points Wnt eluate was subjected to Triton X-114 phase separation. All data are representative of at least two independent experiments with similar results. CM, conditioned medium; WB, western blot; T, total; De, detergent phase; Aq, aqueous phase; s, soluble ECD; Fc, IgG Fc fragment. See also Figures and .

Article Snippet: Anti-mouse GPR124 ECD antiserum was generated by immunizing rabbits (Rockland Immunochemicals, Pottstown, PA) with recombinant mouse GPR124 ECD, which had been cloned, expressed, and purified from stably transfected HEK293 cell conditioned medium as previously described ( ).

Techniques: SDS Page, Transfection, Purification, Affinity Purification, Isolation, Recombinant, Incubation, Western Blot

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: (A) GPR124, RECK, and WNT7A form a ternary complex. HEK293 RECK −/− (control) or HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with the indicated proteins (50 nM) for 72 hr. CM were harvested and subjected to Protein A pull-down. (B) GPR124 does not regulate cell surface RECK:WNT7A complex formation. Cells were transfected with indicated expression constructs for 48 hr. SNAP tags were specifically biotinylated (bSNAP) and cell surface proteins cross-linked in situ by DTSSP (100 μM). Cells were lysed, subjected to streptavidin pull-down, and cross-links reversed. WCL, whole cell lysate. β2AR, β 2 -adrenergic receptor. (C) FZD/GPR124/RECK/WNT7A quaternary complex formation is not detectable in vitro . sRECK-His:WNT7A complex was purified from HEK293 RECK −/− WNT7A CM (72 hr, 100 nM sRECK-His) using Ni-NTA agarose. Indicated purified proteins/sRECK:WNT7A complex (1 μM each) were allowed to form complexes and subjected to Protein A pull-down. Faint non-specific sRECK binding in all lanes. (D) FZD8 elutes WNT7A from RECK. sRECK-Fc:WNT7A complex was isolated from HEK293 RECK −/− WNT7A CM (72 hr, 100 nM sRECK-Fc) by Protein A agarose. Beads were incubated with 0.1, 1, or 10 μM sFZD4/sFZD8 protein (wedges) or glycine, pH 2.9 for 1 hr (eluate 1) and then with 1% SDS (eluate 2). (E) WNT7A binding to RECK and FZD8 is mutually exclusive. HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with indicated proteins (25 nM) for 72 hr. CM were subjected to sequential Ni-NTA agarose (1 st ) and Protein A agarose (2 nd ) pull-downs. (F) RECK dose-dependently enhances FZD8:WNT7A complex formation. HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with sFZD8-His and sRECK-Fc proteins as indicated for 72 hr. CM were subjected to Ni-NTA pull-down. (G) Model of RECK/GPR124-regulated canonical WNT7 signaling. GPR124 ECD binding to RECK strongly enhances RECK/WNT7-induced canonical Wnt signaling by an extracellular mechanism that does not involve intrinsic GPR124 signal transduction or regulation of RECK:WNT7 complex formation. The RECK cystine knot motifs (blue) and cysteine-rich domain (purple) mediate binding of RECK:GPR124 to WNT7, stabilizing WNT7 in its active, monomeric, hydrophobic form. Free WNT7 rapidly converts into an inactive, aggregated, hydrophilic state. The GPR124:RECK complex acts as a stabilizing receptor for WNT7 increasing bioavailability of active, monomeric cell surface WNT7. GPR124:RECK-bound WNT7 is eventually transferred to FZD and LRP5/6 co-receptors by transient direct or indirect interactions. Alternatively, GPR124/RECK/WNT7 could form a stable multi-protein receptor complex with FZD/LRP5/6. All western blots (WB) were performed under reducing conditions. All data are representative of at least two independent experiments with similar results. CM, conditioned medium; s, soluble ECD; Fc, IgG Fc fragment. See also .

Article Snippet: Anti-mouse GPR124 ECD antiserum was generated by immunizing rabbits (Rockland Immunochemicals, Pottstown, PA) with recombinant mouse GPR124 ECD, which had been cloned, expressed, and purified from stably transfected HEK293 cell conditioned medium as previously described ( ).

Techniques: Control, Cell Culture, Transfection, Expressing, Construct, In Situ, In Vitro, Purification, Binding Assay, Isolation, Incubation, Transduction, Western Blot

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse GPR124 ECD antiserum was generated by immunizing rabbits (Rockland Immunochemicals, Pottstown, PA) with recombinant mouse GPR124 ECD, which had been cloned, expressed, and purified from stably transfected HEK293 cell conditioned medium as previously described ( ).

Techniques: Virus, Recombinant, Reporter Assay, Plasmid Preparation, Luciferase, Software, CRISPR