Journal: Chemistry of Materials

Article Title: Advancing Biocapture Substrates via Chemical Lift-Off Lithography

doi: 10.1021/acs.chemmater.7b01970

Figure Lengend Snippet: Figure 4. Native receptor capture. (A) Representative fluorescence image of an L-5-HTP-modified substrate exposed to HEK293 membranes from cells overexpressing 5-HT1A receptors, anti- 5-HT1A receptor primary antibodies, and AlexaFluor® 546 secondary antibodies (excitation at 556 nm). (B) Mean relative fluorescence intensities were significantly different for stamp-noncontact vs. contact regions [t(4)=4,*P<0.05]. Scale bar is 50 µm.

Article Snippet: Human 5-HT1A receptors (0.8 fmol receptor protein/μg membrane protein; 6.4 μg/μL total protein concentration) from transfected human embryonic kidney 293 (HEK293) cells and untransfected HEK293 cell membranes (10 μg/μL total protein concentration) were from Perkin Elmer, Inc. (Waltham, MA, USA).

Techniques: Fluorescence, Modification

Journal: Nature Microbiology

Article Title: Eosinophils enhance granuloma-mediated control of persistent Salmonella infection in mice

doi: 10.1038/s41564-025-02187-1

Figure Lengend Snippet: Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with recombinant EPX (100 ng ml −1 ), MBP (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: In vitro polarization was performed using recombinant mouse MBP or EPX expressed in E. coli (Abbexa abx653285 and Abbexa abx067910) or commercial recombinant human MBP expressed in HEK-293 cells (Elabscience, PKSH032124).

Techniques: Infection, Expressing, Control, Negative Control, Western Blot, Recombinant, Bacteria, Neutralization, Two Tailed Test, MANN-WHITNEY

Journal: Nature Microbiology

Article Title: Eosinophils enhance granuloma-mediated control of persistent Salmonella infection in mice

doi: 10.1038/s41564-025-02187-1

Figure Lengend Snippet: ( a ) Representative immunofluorescence image of MLN tissue sections from mice at 4 w.p.i. showing CD11b (magenta), iNOS (Grey), EPX (Blue) and MBP (Green). Scale bar = 50 μm ( n = 5 mice, 2 sections each). ( b ) S Tm growth in media containing either PBS (Ctrl), EPX (0.155-1.55 μg/ml) or MBP (90-900 ng/ml) for 18 h (n = 3 biological repeats). ( c ) Representative FACS plots showing iNOS and Arginase-1 in MBP (100 ng/ml), EPX (100 ng/ml) MBP and EPX (100 ng/ml each), LPS (100 ng/ml) or IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice. ( d ) Quantification of percentage of iNOS+ and Arginase-1+ cells in MBP (100 ng/ml), EPX (100 ng/ml) MPB and EPX (100 ng/ml each), LPS (100 ng/ml) and IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice (n = BMDMs from 5 mice across two experiments). ( e ) Quantification of iNOS levels by western blots in main Fig. (n = 3 biological repeats). ( f ) Representative western blot showing iNOS expression in murine (mMBP), human (hMBP) (10 ng/ml each) or LPS treated BMDMs from 129×1/SvJ mice (n = 2 biological repeats). ( g ) Quantification of cell death as determined by SYTOX green positive cells (n = 3 biological repeats) ( h ) Frequencies of eosinophils, neutrophils, mast cells and, basophils in the MLN of 129×1/SvJ mice. Basophils are gated as Live, CD45 + CD200R3 + c-Kit − . Mast cells are gated as Live, CD45 + CD200R3 + c-Kit + . Data are presented as Means ± SEM. All analyses are two-tailed and analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction ( b ) or one-way ANOVA ( d , e ). Exact P- values in Supplementary Table . ns = non-significant, * P < 0.05, ** P < 0.01. *** P < 0.001.

Article Snippet: In vitro polarization was performed using recombinant mouse MBP or EPX expressed in E. coli (Abbexa abx653285 and Abbexa abx067910) or commercial recombinant human MBP expressed in HEK-293 cells (Elabscience, PKSH032124).

Techniques: Immunofluorescence, Western Blot, Expressing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: HA-49K orthologs from the A549-based expression system function comparably to natural E3/49K and exhibit a consistent binding activity to CD45 expressing target cells. Cell supernatants containing individual E3/49K variants were incubated together with target cells and bound E3/49K was measured via flow cytometry. Binding activity of HA-49K of HAdV-D64 from the A549-based producer cell line was compared to E3/49K obtained from cells infected with HAdV-D64, HAdV-D64ΔE3 and HAdV-D64ΔE3 + 49K viruses . Supernatants were incubated with wild-type Jurkat (red), Ramos (blue) and the CD45-deficient Jurkat (orange) and Ramos (light blue) cells, respectively. The grey dashed vertical line separates results obtained from transfected cells (left) from those of infected cell lines (right). Binding of HA-49K and E3/49K was detected with 4D1 mAb (A) . Binding activity to Jurkat and Ramos cell lines of recombinant HAdV-D64 HA-49K of was compared with HA-tagged orthologs of HAdV-D8, -D19 and -D36, respectively. Binding was determined with the target cell system as applied in the previous experiment using α-HA Ab (B) . Contrasting the binding of HA-49K and untagged E3/49K from the A549-based expression system to Jurkat cells reveals no negative influence to the binding activity by the HA-tag. Bound E3/49K versions were detected using HA-specific (blue) or E3/49K-specific (4D1, red) mAbs (C) . The binding specificity of HA-49K orthologs was further characterized by competition with untagged E3/49K of HAdV-D64. Residual binding activity of HA-49K orthologs was monitored by flow cytometry using α-HA Abs and a two-step sequential incubation of Jurkat cells with supernatants, containing E3/49K variants. The order of the individual incubations for competition is indicated within the figure. Significant differences to single incubations were analyzed (D) . Cell supernatants from normal A549 cells were utilized as negative controls. The columns represent the mean-MFIs obtained in independent experiments, each depicted as dots, and error-bars represent the standard deviations. Statistical significance (****P<0.0001) was determined via the two-way ANOVA test and is indicated within the panel (D) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Expressing, Binding Assay, Activity Assay, Incubation, Flow Cytometry, Infection, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Activation of Jurkat T cells is inhibited by HA-49K orthologs to an equal extent. Jurkat cells were previously incubated with cell supernatants (red) containing HA-49K orthologs. Cells were washed and stimulation was conducted via receptor cross-linking using immobilized α-CD3 and soluble α-CD28 Abs for 6h. After cell fixation the activation level was determined by flow cytometry-based monitoring of the cell surface expression of the early activation marker CD69. Relative numbers of CD69 positive cells were normalized to CD3/CD28 stimulation control (not shown). Untreated (unstim.) and isotype control (ISO) treated samples were used as negative controls (grey). To show the efficiency of the CD3/CD28 stimulation we treated the cells also with 50 ng/ml Phorbol-12-myristate-13-acetate and 1 µg/ml ionomycin (PMA/Iono, blue). Administration using CD3/CD28 stimulation and unreactive A549 supernatant was utilized to control the effect of the supernatant on CD3/CD28 stimulation (A549, blue) (A) . pErk1/2 levels were identified by immunoblot analysis upon CD3 stimulation of Jurkat cells with 1 µg/ml for 2 min. Sample loading was controlled by detection of β-actin. One representative blot for Jurkat and CD45-/- Jurkat is shown (B) and the relative expression levels of pErk1/2 to β-actin ratios were normalized to the pos. ctrl. (C) . The columns represent the mean of 3 individual experiments (dots), the error-bars represents the standard deviation for A and (C) Statistical differences compared toPMA/ionomycin treatment in (A) and CD3-stimulation in the presence of A549 supernatants in (C) positive controls were analyzed using the two-way ANOVA test. Only significant results were indicated in the figure.

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Activation Assay, Incubation, Flow Cytometry, Expressing, Marker, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Ramos B cell signaling is inhibited by HA-49K orthologs to comparable levels. Ramos B cell signaling was determined to assess the inhibitory potential of HA-49K orthologs in B cells. Previous incubation of Ramos cells with cell supernatants (red) containing various HA-49K orthologs was performed. Unstimulated cells (unstim.) were used as a negative control (grey), co-incubation with unreactive A549 supernatant (A549) served as a positive control indicated in blue. Cells were washed and stimulated via receptor cross-linking using α-λ Abs. The cellular calcium-response was detected by flow cytometry and the mean peak Ca2+-levels (columns) of 3 individual experiments (dots) including standard deviation are shown. Statistical differences compared to A549 were analyzed using two-way ANOVA test (A) . Immunoblot analysis of pErk1/2 levels upon BCR stimulation of Ramos B cell lines with 1 µg/ml α-λ Abs for 2 min. Unstimulated cells (unstim.) served as negative control while α-λ treated cells (pos. crtl.) and co-incubation with A549 supernatant (A549) served as positive controls. Detection of β-actin levels was used as loading control. One representative blot for Ramos and CD45-/- Ramos cells is shown (B) . The relative detection levels of pErk1/2 to β-actin ratios in Ramos cells were normalized to the positive ctrl. The mean (columns) of 3 individual experiments (dots) including standard deviation is shown. Statistical differences to the positive ctrl. were analyzed using the two-way ANOVA test. Only significant results were indicated in the panel (C) . A two-fold dilution series of cell supernatants containing HA-49K-D64 (D) and purified HA-49K-D64 proteins starting at 8 µg per sample (E) was performed. Samples were either supplemented with 0.5 µg per sample hCD45-Fc (+hCD45-Fc, red) or without (hCD45-Fc, blue). After a 1 h incubation period, the binding of HA-49Ks to Ramos cells was detected using α-HA-based flow cytometry. Undiluted supernatant from untransfected A549 cells (A549) or the 8 µg MT protein were utilized as negative controls and shown as single values at the end of the x-axis separated by the grey dashed line. The mean MFI of 3 individual experiments is displayed for each, including standard deviation in the form of error bars. Erk1/2 phosphorylation was analyzed to investigate the prevention of the inhibitory effect HA 49K-D64 by hCD45-Fc receptors via immunoblotting. The supernatant containing HA-49K-D64 proteins was diluted 1:10 and 4 µg purified HA-49K-D64 proteins were incubated for 1 h with 500 ng hCD45-Fc decoy receptors as indicated in the figure. Subsequently, reagents were incubated with Ramos cells for 1 h Cells were lysed after BCR stimulation with 2 µg/ml α-λ Abs for 2 min. Sample loading was controlled by the detection of β-actin levels. One representative blot is presented (F) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Incubation, Negative Control, Positive Control, Flow Cytometry, Standard Deviation, Western Blot, Control, Purification, Binding Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Only HAdV-D infected A549 cells bind soluble hCD45-Fc. A549 cells were infected with HAdV-A12, -B7, -B35, -C5, -D8, -D19, -D36, -D64 and -D64ΔE3 and -E4, viruses with an MOI of 5 for 24 h Efficient infection was confirmed by internal hexon protein expression (red) using 2Hx-2 mAbs in comparison to isotype control staining (grey) of infected A549 cells in flow cytometry. Displayed are representative histograms of productive infections (A) . Infected cells were treated with +/- (red/blue) 0.5 µg hCD45-Fc per sample. Bound CD45 molecules were detected by CD45-ECD staining using α-human pan-CD45 MEM-28 in flow cytometry. Mock infected cells as well as cells infected with HAdV-D64ΔE3 virus served as negative controls. As positive control the cell clone stably expressing HA-49K of HAdV-D64 was applied. The mean of 3 individual experiments (columns) from (dots) including standard deviation, presented as error bars, is shown. Significant differences between +/- hCD45-Fc treatment were determined using the two-way ANOVA test and are indicated in the panel (B) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Infection, Expressing, Comparison, Control, Staining, Flow Cytometry, Virus, Positive Control, Stable Transfection, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Graphical abstract of the putative functional mechanism of E3/49K action in B cells. During BCR antigen ligation, the catalytic activity of CD45 shifts the equilibrium of functional Lyn toward activated Lyn (Lyn Y396). The removal of the inhibitory phosphate group from Y507 sites primes the auto-phosphorylation of Lyn at Y396 sites to induce Lyn kinase activity. Active Lyn promotes phosphorylation of ITAMs and pITAM-attached Syk to facilitate BCR signal transduction. pSyk initiates several signaling pathways, including the MAPK pathway. pErk1/2 and the calcium flux results in transcriptional and cellular activation (A) . E3/49K-mediated dimerization of CD45 molecules prevents the catalytic activity of CD45. As a result, the equilibrium of functional Lyn is shifted toward inactive Lyn (Lyn Y507), increasing the activation threshold during BCR stimulation. As a result of reduced Lyn kinase activity, less pSyk, pErk1/2, and calcium flux are generated, resulting in decreased transcriptional and cellular activation (B) . Dimerization of CD45 by E3/49K may disrupts CD22-CD45 interaction. Active Lyn induces CD22 which enhances its inhibitory effect in reducing BCR signals by affecting pErk1/2 and calcium flux (C) . The figure was created with BioRender.com .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Functional Assay, Ligation, Activity Assay, Transduction, Activation Assay, Generated

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Graphical representation about E3/49K functions. CD45 modulation via binding of E3/49K proteins to its ECD is a common feature of HAdVs of species D. E3/49K ECDs are shed from infected cells and bind to and inhibit CD45 positive target cells. Based on the current hypothesis, inhibition is mediated by enforced dimerization of CD45 , which inhibits leukocyte receptor signaling. B cells are here identified as a new target for E3/49K-mediated immune evasion. Since there are more CD45 expressing leukocytes existing, it is hypothesized that they serve as targets for E3/49K as well. The figure was created with BioRender.com .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Binding Assay, Infection, Inhibition, Expressing

Journal: The FASEB Journal

Article Title: Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF

doi: 10.1096/fj.201900932R

Figure Lengend Snippet: Primer sequences for real-time RT-PCR analysis of RPTKs and PTK7

Article Snippet: The pCMV3-FGFR2-HA, pCMV3-FGFR3-HA, and pCMV3-FGFR4-HA constructs encoding human FGFR2, FGFR3, and FGFR4 with a C-terminal HA-tag, respectively, were purchased from Sino Biological (Beijing, China).

Techniques: Quantitative RT-PCR, Sequencing

Journal: The FASEB Journal

Article Title: Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF

doi: 10.1096/fj.201900932R

Figure Lengend Snippet: Binding of PTK7 to FGFR family members. HA-tagged FGFR1, FGFR2, FGFR3, and FGFR4 (FGFR1-HA, FGFR2-HA, FGFR3-HA, and FGFR4-HA) were immunoprecipitated from HEK293 cells. FLAG-tagged PTK7 (PTK7-FLAG) binding to each FGFR was evaluated by Western blot. An empty vector served as the negative control. GAPDH served as a loading control for total cell lysate blots. The lower panel illustrates quantification of the binding of PTK7 to FGFRs. Each bar represents the mean ± sd from 3 independent experiments.

Article Snippet: The pCMV3-FGFR2-HA, pCMV3-FGFR3-HA, and pCMV3-FGFR4-HA constructs encoding human FGFR2, FGFR3, and FGFR4 with a C-terminal HA-tag, respectively, were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Proteolytic Shedding of Human Colony‐Stimulating Factor 1 Receptor and its implication

doi: 10.1111/jcmm.16474

Figure Lengend Snippet: Cleaved fragments of CSF1R were detected in cell lysate and the medium. A, Sketch of the full length of CSF1R, N‐terminal fragment of CSF1R and full length of TREM2 plasmid. B, HEK293T cells were transfected with pEGFP‐N1 (Vector), TREM2‐EGFP or HA‐CSF1R‐EGFP. After 24 hours, cells were harvested and different fragments of CSF1R were detected by Western blotting with anti‐GFP antibody. The CSF1R‐precursor (~110 kDa), the full length of CSF1R (CSF1R‐FL, ~170 kDa) and the C‐terminal fragment of CSF1R (CSF1R‐CTF, ~ 75 kDa) were detected for CSF1R in cell lysate represent different fractions. Vector is a negative control and TREM2‐EGFP is a positive control. C, HEK293T cells transfected with pEGFP‐N1 (Vector), CSF1R‐NTF‐HA or HA‐CSF1R‐EGFP were cultured with or without 10 μM Batimastat in serum‐free DMEM for 16 hours. The extracellular domain of CSF1R (CSF1R‐ECD) was detected by Western blotting with anti‐HA antibody. D, Bar charts show the relative levels of CSF1R‐ECD (n = 3). CSF1R‐ECD is significantly reduced in the Batimastat group when compared to that in control group. Vector is the negative control and CSF1R‐NTF‐HA is a positive control (n = 3). ***, P <.001, ns, not significant. E, HEK293T cells transfected with pEGFP‐N1 (Vector), TREM2‐EGFP or HA‐CSF1R‐EGFP were incubated with or without 10 μM DAPT for 16 hours. The full‐length and C‐terminal fragments of CSF1R and TREM2 were detected by Western blotting with anti‐GFP antibody. F, Error bars indicate SEM of at least three independent experiments. The CSF1R‐CTF was notably increased in the DAPT group compared to that in control group. Vector is a negative control, and TREM2‐EGFP is a positive control (n ≥ 3). Statistical analysis was done by t test. *, P <.05

Article Snippet: The full‐length CSF1R in HA‐CSF1R‐EGFP was obtained from the 972 amino acid of CSF1R in pCMV3‐CSF1R‐HA (Sino Biological, Beijing, China).

Techniques: Plasmid Preparation, Transfection, Western Blot, Negative Control, Positive Control, Cell Culture, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Proteolytic Shedding of Human Colony‐Stimulating Factor 1 Receptor and its implication

doi: 10.1111/jcmm.16474

Figure Lengend Snippet: Decreased cleavage fragments of disease‐associated CSF1R I794T variant. A, Sketch of the wild‐type (WT) of CSF1R and CSF1R I794T mutant plasmid. B, HEK293T cells were transfected with pCMV3.1‐HA (Vector), CSF1R‐HA (CSF1R‐WT) or CSF1R‐I794T‐HA. Cell lysates were collected and detected by Western blotting with anti‐CSF1R (epitope 11 aa ~ 310 aa; epitope 955 aa ~ 972 aa) or HA‐tag antibody. C, A dramatically reduction of the ECD ratio to the full length (FL) of CSF1R I794T in the medium detected by Western blotting. D, The FL of CSF1R I794T was significantly decreased compared to that of WT CSF1R. E, The ratio of CTF to FL of CSF1R I794T was significantly decreased compared to that of WT CSF1R. F, A dramatically reduction of the secreted ECD of CSF1R I794T in the medium detected by ELISA assay. G, Schematic shedding of CSF1R. CSF1R undergoes a proteolytic cleavage similar to that of TREM2. Extracellular of the CSF1R ectodomain can be shed by MMP or ADAM family and the intramembrane of the CSF1R C‐terminal fragment can be subsequently cleaved by γ‐secretase. H, Schematic shedding of CSF1R I794T variant. The cleaved fragments of disease‐associated CSF1R I794T, underlying the white matter disease of ALSP, were significantly decreased. The released CSF1R ectodomain (ECD) may be proposed as a diagnostic biomarker for ALSP. Whether the released CSF1R intercellular domain (ICD) translocates into the nucleus remains elusive. Error bars indicate SEM of at least three independent experiments. Statistical analysis was done by t test (n = 3). *, P <.05, **, P <.01, ***, P <.001

Article Snippet: The full‐length CSF1R in HA‐CSF1R‐EGFP was obtained from the 972 amino acid of CSF1R in pCMV3‐CSF1R‐HA (Sino Biological, Beijing, China).

Techniques: Variant Assay, Mutagenesis, Plasmid Preparation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Biomarker Assay