Journal: Scientific Reports

Article Title: CX3CL1 homo-oligomerization drives cell-to-cell adherence

doi: 10.1038/s41598-020-65988-w

Figure Lengend Snippet: Electrophoresis of the CX3CL1-YFP fusion protein. ( A ) Cell lysate of L CX3CL1 cells stably transfected with CX3CL1-EYFP was analyzed by SDS-PAGE and Western blot (full length blot in Fig. ). ( B ) Cell lysate (40 µg of total proteins) of L CX3CL1 was analyzed by native electrophoresis using Nu-Page gel in the presence of 1% DDM (dodecylmaltoside) and Western blot. ( C ) The affinity-purified CX3CL1 from L CX3CL1 membranes was analyzed by native PAGE. CX3CL1 was solubilized using CALX173ACE and affinity purified (CX3CL1 antibody). Mini-Protean TGX gel electrophoresis (BioRad) was used for Native PAGE.

Article Snippet: Human CX3CL1 (Chemokine Domain) and polyclonal goat anti-CX3CL1 antibody (clone AF365) were purchased from Biotechne (Lille, France).

Techniques: Electrophoresis, Stable Transfection, Transfection, SDS Page, Western Blot, Affinity Purification, Clear Native PAGE, Nucleic Acid Electrophoresis

Journal: Scientific Reports

Article Title: CX3CL1 homo-oligomerization drives cell-to-cell adherence

doi: 10.1038/s41598-020-65988-w

Figure Lengend Snippet: Single–particle fluorescence analysis of CX3CL1. ( A ) Fluorescence of the membrane preparation of L CX3CL1 diluted in DOPC analyzed by TIRF. Bar 10 µm. ( B ) Examples of the fluorescence of particles tracked during one or two minutes. ( C ) Distribution of the fluorescence step amplitude according to the step number per particle. The grey bars represent the mean of the step amplitude in each case. ( D ) Distribution of the number of elementary fluorescence units per particle calculated after analysis of the fluorescence kinetic of 126 different particles. The Gaussian curve fits the data with an amplitude of 27.5, a mean of 4.3 and a standard deviation of 1.8.

Article Snippet: Human CX3CL1 (Chemokine Domain) and polyclonal goat anti-CX3CL1 antibody (clone AF365) were purchased from Biotechne (Lille, France).

Techniques: Single Particle, Fluorescence, Membrane, Standard Deviation

Journal: Scientific Reports

Article Title: CX3CL1 homo-oligomerization drives cell-to-cell adherence

doi: 10.1038/s41598-020-65988-w

Figure Lengend Snippet: Diffusion rate of CX3CL1 transmembrane peptides analyzed by FRAP and FRAPP. ( A ) The fluorescence kinetics of Giant Unilamellar Vesicles containing either TM24-FITC (filled squares) or SC24-FITC (empty squares) were analyzed after bleaching by circles of various diameter (1, 2, 5 and 10 µm). The recovery constant time was reported versus the bleached area. Each measurement was the mean of triplicates. The slopes of the linear fit are 2.63 and 29.93 for TM24 and SCR24 respectively. ( B )The diffusion rates were calculated based on the mean ± SEM of 12 measurements data used to give the ( A ). ( C ) The fluorescence kinetics of lipidic cubic “sponge” phase containing either TM24-FITC (filled squares) or SC24-FITC (empty squares) were analyzed after bleaching by interference pattern with various interfringe (20, 27, 46 and 58 µm). The recovery constant time was reported versus the interfringe distance. Each measurement is the mean of triplicates. The slopes of the linear fit are 252 ± 10 and 1093 ± 144 for TM24 and SCR24, respectively. ( D ) The diffusion rates were calculated based on the mean ± SEM of 12 measurements data used to give the ( C) . Note that the diffusion rate appeared dramatically higher in sponge phase since the fluorescent molecules could move in tridimensional milieu.

Article Snippet: Human CX3CL1 (Chemokine Domain) and polyclonal goat anti-CX3CL1 antibody (clone AF365) were purchased from Biotechne (Lille, France).

Techniques: Diffusion-based Assay, Fluorescence

Journal: Scientific Reports

Article Title: CX3CL1 homo-oligomerization drives cell-to-cell adherence

doi: 10.1038/s41598-020-65988-w

Figure Lengend Snippet: Diffusion rate in cellular membrane of the CX3CL1 protein in the presence of TM24 and SCR24 peptides. The lateral diffusion rate of the CX3CL1-EYFP protein was assayed after transient expression in COS-7 cell line after 15 min preincubation in the presence or not of 3 or 10 µM of TM24 or SCR24 peptides. Each point is the mean of triplicates ± SD.

Article Snippet: Human CX3CL1 (Chemokine Domain) and polyclonal goat anti-CX3CL1 antibody (clone AF365) were purchased from Biotechne (Lille, France).

Techniques: Diffusion-based Assay, Membrane, Expressing

Journal: Scientific Reports

Article Title: CX3CL1 homo-oligomerization drives cell-to-cell adherence

doi: 10.1038/s41598-020-65988-w

Figure Lengend Snippet: Specific inhibition of the CX3CL1-dependent cell adherence by the peptide TM24. (A ) Real time adherence of CHO CX3CR1 cells to L 929 (dashed traces) or L CX3CL1 cells (solid traces) as assayed by the LigandTracer technique, in the presence of 5 µM TM24 (red traces), 5 µM SCR24 (blue traces) or none (black traces). The data were normalized using the control trace with L CX3CL1 cells in the absence of peptide (100% after 60 minutes). The curves are the mean of three independent experiments. (B ) Specific adherence of CHO CX3CR1 cells to L CX3CL1 cells using data of ( A ). The data obtained with L CX3CL1 cells were subtracted from data obtained with L 929 and normalized at 100% at the 60 minutes time. The bars in the right show the specific adherence after 60 minutes (mean of triplicates ± SD) in the presence of 5 µM TM24 (red) and 5 µM SCR24 (blue). (C ) Real time binding of 100 nM of fluorescent CX3CL1 to coated CHO CX3CR1 cells using the LigandTracer technique, in the presence of 5 µM TM24 (red trace), 1 µM unstained CX3CL1 (dashed trace) or none (black trace). The data were normalized using the control trace without peptide (100% after 60 minutes). (D ) Specific adherence of CHO CX3CR1 cells to L CX3CL1 cells after 60 minutes in the presence of various TM24 peptide concentrations and in the presence (filled squares) or in absence (empty squares) of 1 µg/ml of anti-CX3CL1 antibody. The data were calculated and normalized as in ( B ). Experiments are performed in duplicate except for the 1 µM TM24 concentration done in triplicates (mean ± SD).

Article Snippet: Human CX3CL1 (Chemokine Domain) and polyclonal goat anti-CX3CL1 antibody (clone AF365) were purchased from Biotechne (Lille, France).

Techniques: Inhibition, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: The CX3CL1 oligomerization is required for efficient CX3CR1-specific cell adherence

doi: 10.1101/865998

Figure Lengend Snippet: A.Cell lysate of L CX3CL1 cells stably transfected with CX3CL1-EYFP was analyzed by SDS-PAGE and Western blot. B.Cell lysate of L CX3CL1 was analyzed by native electrophoresis using Nu-Page gel in the presence of 1% DDM (dodecylmaltoside) and Western blot. C. The affinity-purified CX3CL1 from L CX3CL1 membranes was analyzed by native PAGE. CXCL3 was solubilized using CALX173ACE and affinity purified (CXCL1 antibody). Mini-proteon TGX gel electrophoresis (Biorad) were used for Native PAGE.

Article Snippet: The immunodetection of CX3CL1 was performed by using the SNAP i.d. system ( Millipore ) with primary antibody (R&D system, MAB3651) against CX3CL1 (1/500 dilution) and revealed using a mouse HRP secondary antibody (Santa Cruz, 3/10,000 dilution).

Techniques: Stable Transfection, Transfection, SDS Page, Western Blot, Electrophoresis, Affinity Purification, Clear Native PAGE, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: The CX3CL1 oligomerization is required for efficient CX3CR1-specific cell adherence

doi: 10.1101/865998

Figure Lengend Snippet: A. Fluorescence of the membrane preparation of L CX3CL1 diluted in DOPC analyzed by TIRF. Bar 10µm B. Examples of the fluorescence of two particles tracked during two minutes C. Distribution of the number of elementary fluorescence units per particle calculated after analysis of the fluorescence kinetic of 126 different particles. The Gaussian curve fits the data with an amplitude of 27.5, a mean of 4.3 and a standard deviation of 1.8.

Article Snippet: The immunodetection of CX3CL1 was performed by using the SNAP i.d. system ( Millipore ) with primary antibody (R&D system, MAB3651) against CX3CL1 (1/500 dilution) and revealed using a mouse HRP secondary antibody (Santa Cruz, 3/10,000 dilution).

Techniques: Fluorescence, Membrane, Standard Deviation

Journal: bioRxiv

Article Title: The CX3CL1 oligomerization is required for efficient CX3CR1-specific cell adherence

doi: 10.1101/865998

Figure Lengend Snippet: The lateral diffusion rate of the CX3CL1-EYFP protein was assayed after transient expression in COS-7 cell line after 15 min preincubation in the presence or not of 3 or 10 µM of TM24 or SCR24 peptides. Each point is the mean of triplicates±SD.

Article Snippet: The immunodetection of CX3CL1 was performed by using the SNAP i.d. system ( Millipore ) with primary antibody (R&D system, MAB3651) against CX3CL1 (1/500 dilution) and revealed using a mouse HRP secondary antibody (Santa Cruz, 3/10,000 dilution).

Techniques: Diffusion-based Assay, Expressing

Journal: bioRxiv

Article Title: The CX3CL1 oligomerization is required for efficient CX3CR1-specific cell adherence

doi: 10.1101/865998

Figure Lengend Snippet: A. Real time adherence of CHO CX3CR1 cells to L 929 (dashed traces) or L CX3CL1 cells (solid traces) as assayed by the LigandTracer™ technique, in the presence of 5µM TM24 (red traces), 5µM SCR24 (blue traces) or none (black traces). The data were normalized using the control trace with L CX3CL1 cells in the absence of peptide (100% after 60 minutes). The curves are the mean of three independent experiments. B. Specific adherence of CHO CX3CR1 cells to L CX3CL1 cells using data of . The data obtained with L CX3CL1 cells were subtracted from data obtained with L 929 and normalized at 100% at the 60 minutes time. The bars in the right of the Fig shows the specific adherence after 60 minutes (mean of triplicates±SD) in the presence of 5µM TM24 (red) and 5µM SCR24 (blue). C. Real time binding of 100nM of fluorescent CX3CL1 to coated CHO CX3CR1 cells using the LigandTracer™ technique, in the presence of 5µM TM24 (red trace), 1µM unstained CX3CL1 (dashed trace) or none (black trace). The data were normalized using the control trace without peptide (100% after 60 minutes). D. Specific adherence of CHO CX3CR1 cells to L CX3CL1 cells after 60 minutes in the presence of various TM24 peptide concentrations and in the presence (filled squares) or in absence (empty squares) of 1µg/ml of anti-CX3CL1 antibody. The data were calculated and normalized as in . Experiments are performed in duplicate except for the 1µM TM24 concentration done in triplicates (mean±SD).

Article Snippet: The immunodetection of CX3CL1 was performed by using the SNAP i.d. system ( Millipore ) with primary antibody (R&D system, MAB3651) against CX3CL1 (1/500 dilution) and revealed using a mouse HRP secondary antibody (Santa Cruz, 3/10,000 dilution).

Techniques: Control, Binding Assay, Concentration Assay