Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Western blot showing knockdown of endogenous RHBDL2 protein in HaCaT cells by multiple shRNAs (01, 00, 02) compared with wild-type (WT) and vector control (V) cells. B. Statistics of the duplicate proteomics experiments. Numbers of identified and quantified proteins ranked by their topology are shown for each of the SILAC experiments, their overlap and union. Type I membrane proteins (with a signal peptide and a single transmembrane helix), which are potential rhomboid substrates, represent about 25% of the secretome in each case and are shown in pale green. Blue, type II membrane proteins; turquoise, polytopic transmembrane proteins; grey, secreted proteins; black, intracellular proteins. C. Changes in membrane protein abundance in HaCaT keratinocyte secretome induced by RHBDL2 expression. In two independent reverse experiments, WT and R2kd HaCaT cells were isotopically labelled by heavy or light lysine and arginine, the media from both populations were pooled and the lectin-enriched glycoproteins were identified and quantified by MS analysis. The abundance ratios of all transmembrane proteins identified in both experiments (i.e. the overlap of the two datasets) were plotted against each other. Two-fold enrichment was set as a significance threshold (dotted line). Membrane proteins occurring in the grey quadrant showed consistent enrichment in both experiments and represent strong candidates for RHBDL2 substrates. D. HaCaT cells were stably transfected with constructs encoding fluorescent fusions of EGFR (GFP) and RHBDL2 (mCherry) and analysed by confocal microscopy. Scale bar = 10 µm. E. Media from WT, vector control (V) and RHBDL2 knockdown (R2kd) HaCaT and MDA-MB-468 cells was concentrated and probed with an antibody raised against the EGFR ectodomain to detect RHBDL2 dependent shedding at endogenous levels of expression. F. Conditioned media from HaCaT cells were divided equally and one half was subjected to high-speed ultracentrifugation (UC) to remove membranes including exosomes. The supernatant after ultracentrifugation and the untreated medium were immunoblotted using separate primary antibodies raised against the extracellular N-terminal (NT) or the intracellular C-terminal part (CT) of EGFR. Ultracentrifugation selectively depletes the full-length form of EGFR, which is reactive against the C-terminal antibody. G . Contribution of metalloproteases to EGFR shedding. N-terminally Strep-tagged EGFR was expressed in HEK293ET cells alongside HA-tagged RHBDL2 or a catalytically inactive mutant (S187A) and cultivated for 24 hrs in the presence or absence of 10 µM BB94. H. The candidate cleavage sites were identified by mass spectrometry (MS) of the purified ectodomain ( Fig. S1 ). Candidate P1 residues were mutated to proline to produce uncleavable mutants and tested by co-overexpression with the WT or inactive mutant enzyme (S187A) in HEK293ET cells. I. An RHBDL2 dependent C-terminal EGFR fragment (open arrow) can be produced by overexpression of the wild-type enzyme in HaCaT cells, but not the S187T inactive mutant (left). RHBDL2 overexpressing cells were incubated overnight with 10 nM PR-171 (PR), 10 µM lactacystin (LC), 10 µM chloroquine (CQ) or 100 nM bafilomycin A1 (BA1) to determine the fate of the fragment (middle). The fragment can also be observed by overnight treatment with bafilomycin A1 at endogenous levels of RHBDL2 expression (right).

Article Snippet: Recombinant EGFR ectodomain was purchased from Sinobiological, Inc. (cat. no. 10001-H02H-50).

Techniques: Western Blot, Knockdown, Plasmid Preparation, Control, Multiplex sample analysis, Membrane, Quantitative Proteomics, Expressing, Stable Transfection, Transfection, Construct, Confocal Microscopy, Mutagenesis, Mass Spectrometry, Purification, Over Expression, Produced, Incubation

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A-C . HaCaT cell lines were seeded equally and grown to confluent monolayers over 48 h. Cells were lysed on ice in the presence of protease and phosphatase inhibitors and samples were diluted to equal concentrations of total protein. Lysates were then probed for various components of EGFR signalling pathways by western blotting. D. HaCaT cell migration was analysed by time-lapse phase-contrast microscopy over the indicated timeframe. Left panel shows the time sequence of the cell colony outline during spreading of wild-type, R2kd and vector transfected cells. Colony outlines were superimposed from first image to last image. For clarity, outlines corresponding to initial 340 min are shown in 20 min increments. Right panel shows quantification of average distance migrated after 16 h. Asterisks correspond to P values (ns=P > 0.05, *=P ≤ 0.05,**= P ≤ 0.01, ***=P ≤ 0.001 and ****= P ≤ 0.0001). E. Gap-closure assay. WT, R2kd and vector HaCaT cells were grown to confluence around removable silicone inserts. After insert removal, gap closure was observed by time-lapse microscopy over 16 h. The average distance migrated is quantified in the box and whisker plot. F. Quantification of migration of WT-HaCaT, R2kd cells and rescue cells in which shRNA refractory wild type RHBDL2 (R2kd+WT) or its inactive S187T mutant (R2kd+mut) were re-introduced into R2kd cells. RHBDL2 overexpression was induced by 5 µg/mL cumate where indicated. G. Depletion of RHBDL2 potentiates invasion of HaCaT cells into a 3D collagen matrix. Spheroids of HaCaT keratinocytes, wild-type (WT), Vector control (V), RHBDL2 knockdown (R2kd) and rescue cells (R2kd+WT, R2kd+mut) were embedded in collagen and their invasion was measured after 72 h by comparing the total area of invaded cells relative to the area of the cell spheroid at 0 h. Invasion is quantified from 5-8 spheroids in each of 3 replicate experiments (total ≥20). Statistical analyses by Tukey’s multiple comparisons test were performed using Prism software (GraphPad Software Inc.). H. HaCaT cell proliferation rate after 48 h growth in 3D collagen was assayed using the Alamar Blue assay. The level of fluorescence is proportional to the metabolic activity and hence can be used to estimate the number of cells relative to each line. The box and whisker plot is generated from 4 replicate experiments. Statistical analyses by Tukey’s multiple comparisons test were performed using Prism software (GraphPad Software Inc.).

Article Snippet: Recombinant EGFR ectodomain was purchased from Sinobiological, Inc. (cat. no. 10001-H02H-50).

Techniques: Western Blot, Migration, Microscopy, Sequencing, Plasmid Preparation, Transfection, Time-lapse Microscopy, Whisker Assay, shRNA, Mutagenesis, Over Expression, Control, Knockdown, Software, Alamar Blue Assay, Fluorescence, Activity Assay, Generated

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Quantification of distance migrated by R2kd cells treated with 1 µM AG1478 compared with untreated WT and R2kd cells. B. Quantification of migration of R2kd cells treated with 10 µM GM6001 or BB94 compared with untreated R2kd cells. C. Distance migrated by R2kd cells incubated with conditioned media from WT cells compared with untreated WT, vector and R2kd cells. D. Distance migrated by WT and R2kd cells treated with recombinant EGFR ectodomain (ED, 1 µg/mL), a cocktail of EGFR ligands (EGFRL; EGF, TGFα and Amphiregulin, 10 ng/ml each) or both. E. Distance migrated by WT cells treated with conditioned media from R2kd cells. Where indicated, conditioned media were obtained from R2kd cells treated overnight with 10 µM BB94 or pre-treated with EGFR ectodomain for 1 hour prior to exchange. F. Cell surface levels of endogenous EGFR and CD138 were analysed in WT (green) and R2kd (magenta) HaCaT cells. Intact cells were stained on ice with EGFR and CD138 antibodies, or with rabbit IgG and secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of three biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. Asterisks correspond to P values (ns=P > 0.05, *=P ≤ 0.05,**= P ≤ 0.01, ***=P ≤ 0.001 and ****= P ≤ 0.0001).

Article Snippet: Recombinant EGFR ectodomain was purchased from Sinobiological, Inc. (cat. no. 10001-H02H-50).

Techniques: Migration, Incubation, Plasmid Preparation, Recombinant, Staining, Control, Immunostaining, Flow Cytometry, Fluorescence, Software

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. HaCaT cells were incubated overnight in serum free media -/+ 10 µM BB94 and 10 ng/mL EGF or TGFα. The media and lysates were harvested and immunoblotted for EGFR. Shedding was quantified using Li-Cor software to determine signal intensity in the media and is displayed relative to the untreated sample above the media panel. B. HaCaT cells stably expressing EGFR-eGFP and mCherry-RHBDL2 were incubated with the indicated EGFR ligand at 10 ng/mL on ice for 1 h, then washed and returned to 37 °C for 15 min and fixed with 3% PFA. GFP and mCherry fluorescence was imaged using confocal microscopy. Scale bar= 10 µm C. HaCaT cells were incubated overnight in serum free media -/+ 10 µM BB94, 80 µM Dynasore and 10 ng/mL EGF. The media and lysates were harvested and immunoblotted for EGFR. Shedding was quantified using Li-Cor software to determine signal intensity in the media and is displayed relative to the untreated sample above the media panel. D. HaCaT-vector (V) and R2kd cells were incubated in serum-free media -/+ 1 µM ionomycin (IM) for 2 h, media and lysates were harvested and immunoblotted for RHBDL2 substrates. E . Stimulation of RHBDL2-mediated EGFR shedding by calcium ionophore treatment requires extracellular calcium. HaCaT cells were incubated for 2 h in serum free media -/+ ionomycin (1 µM) and EGTA (2 mM), BAPTA (2 mM) or Calcium free PBS. A 40 min pre-treatment with BAPTA-AM (100 µM) was performed prior to washing with PBS and subsequent incubation with calcium containing serum-free medium containing ionomycin (1 µM). F. Live HaCaT cells stably overexpressing EGFR-GFP (green) and mCherry-RHBDL2 (red) were treated with 1 µM ionomycin and imaged at regular intervals for 2 h following treatment at 37 °C. Arrowheads indicate sites of plasma membrane blebbing. Scale bar = 5 µm. G. Activation of PLC promotes EGFR shedding. HaCaT cells were incubated for 2 h in serum free media containing ionomycin or increasing concentrations of m-3M3FBS (left). The media and lysates were harvested and immunoblotted for EGFR. In a second experiment, cells were incubated in the presence or absence of m-3M3FBS, the media and lysates were harvested in buffer containing phosphatase inhibitors and immunoblotted for EGFR, PLCγ and phospho-PLCγ (right). H . HaCaT control (V) and R2kd cells were incubated for 2 h in serum-free media -/+ 100 µM m-3M3FBS then media and lysates were harvested and immunoblotted for EGFR. I . HaCaT cells were treated for 2 h with 80 µM m-3M3FBS in serum-free media -/+ calcium. Media and lysates were harvested and immunoblotted for EGFR. UT, untransfected

Article Snippet: Recombinant EGFR ectodomain was purchased from Sinobiological, Inc. (cat. no. 10001-H02H-50).

Techniques: Incubation, Software, Stable Transfection, Expressing, Fluorescence, Confocal Microscopy, Plasmid Preparation, Clinical Proteomics, Membrane, Activation Assay, Control

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. RHBDL2 mRNA comparison by qPCR in the Ker-CT and HaCaT cells. Gene expression was normalized to GAPDH and the level of RHBDL2 expression in HaCaT cells was used to normalize RHBDL2 expression in all other cell lines, also in panel F. The mRNA analysis was done from three biological replicates, each in three technical replicates. Error bars show standard deviation. B. and C. Immunoblotting of conditioned medium and lysate after 4 h incubation with 5 mM calcium and 1 µM ionomycin detecting shedding of EGFR in primary NHEK-Ad and immortalized keratinocytes Ker-CT that is inhibited by a RHBDL2 ketoamide inhibitor compound 11 (50 µM) confirming RHBDL2 dependency. Tubulin is was used as a loading control. D. Immunoblotting of conditioned medium after 4 h incubation of Ker-CT keratinocytes with 100 µM PLCγ activator m-3M3FBS in the absence and presence of RHBDL2 inhibitor compound 11 (50 µM). E. Immunoblotting of conditioned medium after 4 h incubation of Ker-CT with thapsigargin (2 µM), bradykinin (10 µM) or 5 mM calcium as a positive control that induces RHBDL2 dependent EGFR shedding. F. RHBDL2 mRNA detection by qPCR in the N/TERT keratinocyte derived RHBDL2 knockout (R2ko) cell lines with comparison to the HaCaT and HaCaT RHBDL2 knockdown (R2kd) cells. The mRNA analysis was done from three biological replicates, each in three technical replicates. Error bars show standard deviation. G. Immunoblotting of conditioned medium and lysate of N/TERT keratinocytes to confirm RHBDL2 dependency of EGFR shedding in these cells and to validate the RHBDL2 KO (CB and CC) generated in N/TERT keratinocytes. Constitutive (48 h) or calcium (5 mM) stimulated (4 h) shedding of EGFR is inhibited by compound 11 (50 µM) in the WT, confirming its RHBDL2 dependence.

Article Snippet: Recombinant EGFR ectodomain was purchased from Sinobiological, Inc. (cat. no. 10001-H02H-50).

Techniques: Comparison, Gene Expression, Expressing, Standard Deviation, Western Blot, Incubation, Control, Positive Control, Derivative Assay, Knock-Out, Knockdown, Generated

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Constitutive activity of RHBDL2. Monomeric EGFR (green) is cleaved by RHBDL2 (blue) and the released EGFR ectodomain binds EGFR ligands (yellow) in the pericellular space, titrating down their free pool. Dimeric (activated) EGFR is protected from cleavage by RHBDL2. The remaining membrane and cytosolic domains of EGFR are endocytosed and degraded in the lysosome. B . Protracted elevation of intracellular calcium increases RHBDL2 activity and shedding of EGFR ectodomain about 10-fold, which titrates down extracellular ligand levels and limits surface levels of EGFR. C. Loss of RHBDL2 activity leads to increased EGFR signalling, as there are no EGFR ectodomain decoys released that would titrate down the EGFR ligands.

Article Snippet: Recombinant EGFR ectodomain was purchased from Sinobiological, Inc. (cat. no. 10001-H02H-50).

Techniques: Activity Assay, Membrane

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 2. Representative normalized PL intensity decay curves (measured at 659 ± 10 nm) of AB−QD and Tb−AB conjugates mixed with increasing concentrations of EGFR (0 nM, black; 0.9 nM, red; 2.25 nM, blue; 4.5 nM, orange; and 9 nM, green) for the Tb-Cet/Mat-QD650 (A), Tb-Cet/MatFab-QD650 (B), and Tb-EgA1/EgB4-QD650 (C) FRET pairs. Yellow arrows indicate QD FRET sensitization with increasing EGFR concentration. The magenta curves present the mathematical sum of only AB-QD650 and only the Tb−AB conjugate. Although the optical bandpass filters (cf. Figure 1B) were selected to minimize Tb crosstalk into the QD detection channels, there was still significant Tb PL detectable. For intensity normalization, all curves within one graph where multiplied by the value that led to unity intensity for the magenta curve at 0.5 ms. PL decay curves (Tb donors and QD acceptors) of all immunoassays can be found in the Supporting Information (Supporting Figures S1−S8).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Concentration Assay

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 3. Homogeneous FRET immunoassay calibration curves against EGFR using different Tb−AB and AB−QD conjugates (blue, Tb-Cet/MatFab-QD605; green, Tb-Cet/MatFab-QD650; cyan, Tb- Cet/Mat-QD605; black, Tb-Mat/Cet-QD650; red, Tb-Cet/Mat- QD650; orange, Tb-EgA1/EgB4-QD650; brown, Tb-EgA1/EgB4- QD605; magenta, Tb-Cet/MatFab-QD650 measured in serum samples). [EGFR] corresponds to the variable EGFR dimer concentrations (recombinant human EGFR Fc chimera) in the 50 μL EGFR samples (in buffer or serum), whereas the overall measuring volume of 150 μL also contained 100 μL of a constant assay solution (50 μL of Tb−AB conjugate with 9 nM AB and 50 μL of AB−QD conjugate with 1.5 nM of QD650 or 3 nM of QD605 for all samples). Individual curves for the Tb and QD signals (that lead to the calculation of FR) and for the determination of LODs can be found in the Supporting Information (Supporting Figures S11−S18).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Recombinant

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 5. Specificity of the different AB pairs for EGFR (red) or HER2 (blue). Tb-Cet/Mat-QD650 (A) and Tb-Tras/Pert-QD650 (B) showed a concentration-dependent FRET-ratio increase only for their respective receptors. Tb-Pert/Mat-QD650 (C) and Tb-Tras/Mat-QD650 (D) showed a concentration-dependent FRET-ratio increase for HER2, which indicates a cross-reactivity of the EGFR-specific Mat to HER2. Tb-Cet/Pert-QD650 did not lead to any concentration dependent FRET-ratio increase (Supporting Figure S27).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Concentration Assay

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 6. Duplexed immunoassay (all samples containing Tb-Cet, Tb-Tras, Mat-QD605, and Pert-QD650) calibration curves of EGFR (FRET-ratio of QD605 and Tb PL intensities, A) and HER2 (FRET-ratio of QD650 and Tb PL intensities, B) for increasing (0 nM, black; 0.3 nM, green; 0.6 nM, magenta; 1.2 nM, red; 1.8 nM, cyan; 2.4 nM, brown; 3 nM, blue) HER2 (A) and EGFR (B) concentrations.

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques:

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 7. Duplexed FRET immunoassay for the quantification of EGFR (blue squares) and HER2 (red dots) within 19 different samples. Dotted lines represent the known concentrations, and data points represent the measured concentrations (±10% error bars).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques:

Journal: Biomedicines

Article Title: A Rational Designed Novel Bispecific Antibody for the Treatment of GBM

doi: 10.3390/biomedicines9060640

Figure Lengend Snippet: ( a ) EGFRvIII antigen expression level on mutant GBM cell line U87MG.ΔEGFR and wild-type GBM cell line U87MG. ( b ) A binding activity comparison of EGFRvIII-BsAb and CD3 mAb with Jurkat cells (CD3-positive) (upper), as well as a binding activity comparison of the EGFRvIII-BsAb and the EGFRvIII mAb with U87MG.ΔEGFR cells (EGFRvIII-positive) (lower). ( c ) Photographs of the redirection of T cells to cancer cells by 0.01 ng/mL EGFRvIII-BsAb or EGFRvIII mAb. ( d ) FACS analysis of the redirection of CD3+ Jurkat cells to cancer cells by EGFRvIII-BsAb. Jurkat (CD3+) cells labeled by PKH26 (PE-A), as well as U87MG.ΔEGFR cells labeled by CFSE (FITC-A).

Article Snippet: The EGFRvIII antigen (AVI10494; R&D System, Minneapolis, MN, USA) and extracellular domain of human CD3D/CD3E heterodimer (CT038-H2508H; Sino Biological, Beijing, China) were immobilized to a CM5 chip (29149603; GE Healthcare, Chicago, IL, USA) surface using standard protocols with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) amine.

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Comparison, Labeling

Journal: Cell reports

Article Title: Granulocyte colony stimulating factor promotes scarless tissue regeneration

doi: 10.1016/j.celrep.2024.114742

Figure Lengend Snippet: (A) Parabiosis between WT:WT ( n = 4, red solid line), CXCR2 KO:CXCR2 KO ( n = 3, blue dotted line), and WT:CXCR2 KO mice ( n = 4, black dotted line). Shown is the percentage of ear hole closure. 2-way ANOVA comparing WT:KO pairs to WT:WT pairs. (B) ELISA measuring cytokine expression in injured WT ( n = 4 for day 3, n = 3 for day 7) and CXCR2 KO ( n = 6 for day 3, n = 3 for day 7) plasma. Unpaired two-tailed Student’s t test. (C) WT ( n = 6), CXCR2 KO ( n = 7) and G-CSF depleted CXCR2 KO ( n = 4) plasma was injected daily into the wound bed of WT mice undergoing WIHN for the first 3 days after injury. Shown are representative photographs and quantification of hair follicles. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (D) G-CSF ( n = 9) or PBS (control, n = 7 ) was injected daily into the wound bed of WT mice undergoing WIHN for the first 3 days after injury. Representative photographs of whole-mount and scanning electron microscopy demonstrating unpigmented hairs in the center of the healed areas. Right: quantification of hair follicles. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (E) Representative immunofluorescence images of PBS and G-CSF-injected WT wound beds depicting hair follicle structures (Krt14+, Krt6+) Scale bars, 100 μM. (F) Representative photographs and quantification of scar size of G-CSF-treated ( n = 8) or PBS-treated (control, n = 3 ) stented back wounds at day 28 after injury. Scale bars, 1 mm. Unpaired two-tailed Student’s t test. (G) Representative trichrome-stained tissue sections from G-CSF- or PBS-treated stented back wounds. A black line highlights scar size. (H) Quantification of scar diameter for G-CSF ( n = 6) or PBS-treated ( n = 3) mice. Unpaired two-tailed Student’s t test. (I) Wound fibrosis assessed by picrosirius red staining in G-CSF-treated ( n = 14 sections) or PBS-treated ( n = 10 sections). Unpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Mean ± SEM are plotted.

Article Snippet: G-CSF , Med Chem Express , HY-P70608.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Clinical Proteomics, Two Tailed Test, Injection, Control, Electron Microscopy, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Granulocyte colony stimulating factor promotes scarless tissue regeneration

doi: 10.1016/j.celrep.2024.114742

Figure Lengend Snippet: (A) Dot plot demonstrating average expression and percentage of immune cells expressing Csf3r . (B) Analysis of key cell-to-cell interactions between immune cells in the ear skin of WT (salmon color) and CXCR2 KO (blue color) mice. Mac, macrophage; T, T cell. (C) Dot plot demonstrating average gene expression of key genes between WT and CXCR2 KO macrophages. (D) GSEA of macrophage populations in WT and CXCR2 KO wounded skin. (E) Representative images and quantification of immunofluorescence of WT and CXCR2 KO wounded skin for CD80 ( n = 4), COX2 ( n = 4), CD163 ( n = 12 for WT and n = 11 for KO), MRC1 ( n = 10 for WT and n = 6 for KO), and ARG1 ( n = 10 for WT and n = 6 for KO). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (F) Representative H&E immunostaining images and quantification of PBS- and G-CSF-injected stented back wounds of WT mice for CD163 ( n = 7 for PBS and n = 5 for G-CSF), MRC1 ( n = 5 for PBS and n = 7 for G-CSF), CD31 ( n = 8 for PBS and n = 7 for G-CSF) and Ki67 ( n = 5 for PBS and n = 4 for G-CSF). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.01. Mean ± SEM are plotted.

Article Snippet: G-CSF , Med Chem Express , HY-P70608.

Techniques: Expressing, Gene Expression, Immunofluorescence, Two Tailed Test, Immunostaining, Injection

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Screening flow to identify novel Abs from patients with esophageal cancer who had survived more than 5 years after recurrence. EGFR, epidermal growth factor receptor; SCC, squamous cell carcinoma

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques:

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Identification of antigens recognized by KT112 Abs. (A) Results of SDS‐PAGE after immunoprecipitation. Lane 1, OE21_membrane fraction; lane 2, OE21_supernatant fraction; lane 3, OE21_wash fraction; lane 4, OE21_elute fraction; lane 5, MRC5_elute fraction; lane 6, MRC5_wash fraction; and lane 7, MRC5_supernatant fraction. (B) Reactivity of the cell after siRNA treatment by flow cytometry analysis. Thin solid line shows the reactivity of the DNP Ab to cells treated with negative control siRNA, the thick solid line shows the reactivity of the KT112 Ab to cells treated with negative control siRNA, and the dotted line shows the reactivity of KT112 Abs to cells treated with epidermal growth factor receptor (EGFR) siRNA. vIII, variant III; scFv, single‐chain Fv

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques: SDS Page, Immunoprecipitation, Membrane, Flow Cytometry, Negative Control, Variant Assay

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Reactivity to recombinant epidermal growth factor receptor (EGFR; left panel) and EGFR variant III (vIII; right panel) was analyzed by ELISA. Black circles, KT112 Ab; black squares, cetuximab; black triangles, AM1; white circles, DNP Ab (5 μg/ml only); white triangles, panitumumab

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques: Recombinant, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Competitive inhibition of each epidermal growth factor receptor (EGFR) Ab. The reactivity of each EGFR Ab to OE21 cells was analyzed by flow cytometry. The horizontal axis indicates the Ab name, and the vertical axis indicates mean fluorescence intensity. (A) Reactivity of the KT112 Ab in the presence of a competitive Ab is shown. Concentrations of the competitive Abs are 0.3, 1, 3, 10, and 30 μg/ml. 2,4‐Dinitrophenol (DNP) Ab was analyzed only at 30 μg/ml. AM1 Ab was analyzed at 3, 10, and 30 μg/ml. The concentration of the biotinylated KT112 Ab for detection is 0.8 μg/ml. (B) Reactivity of each Ab in the presence of the KT112 Ab. Concentrations of the competitive Abs are 0.3, 1, 3, 10, and 30 μg/ml. Concentrations of the Abs for detection are 0.8 μg/ml for the biotinylated KT112 Ab, 0.1 μg/ml each for biotinylated cetuximab and panitumumab, and 2 μg/ml for the biotinylated AM1 Ab

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques: Inhibition, Flow Cytometry, Fluorescence, Concentration Assay