recombinant human egfr protein  (TargetMol)

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: Drug Design, Development and Therapy

Article Title: Circulating Butyrate Attenuates Cetuximab Efficacy in Colorectal Cancer Through EGFR and AMPK–Wip1 Signaling

doi: 10.2147/DDDT.S574116

Figure Lengend Snippet: NaBu enhances EGFR signaling in KRAS-WT CRC cells. ( A ) Human phospho-kinase array of Sw48 cells treated with NaBu, showing increased phosphorylation of EGFR, AKT, and ERK1/2, and decreased phosphorylation of p53 and p38. ( B ) Heatmap representation of differential phosphorylation. ( C ) Western blot validation of p-EGFR, p-AKT, and p-ERK1/2 in KRAS-WT cells (Caco2, Sw48) treated with NaBu ± CTX. ( D ) Microscale thermophoresis (MST) analysis of EGF–EGFR binding in the presence or absence of NaBu. Enhanced binding in the presence of NaBu is reflected by a leftward shift of the binding curve and a reduced apparent dissociation constant. The inset shows representative MST time traces used for curve fitting and binding analysis. NaBu (+), presence of sodium butyrate; NaBu (−), absence of sodium butyrate.

Article Snippet: Recombinant human EGFR protein (TargetMol) was fluorescently labeled with Alexa FluorTM 647 NHS Ester (A37573, Invitrogen) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Western Blot, Biomarker Discovery, Microscale Thermophoresis, Binding Assay

Journal: Drug Design, Development and Therapy

Article Title: Circulating Butyrate Attenuates Cetuximab Efficacy in Colorectal Cancer Through EGFR and AMPK–Wip1 Signaling

doi: 10.2147/DDDT.S574116

Figure Lengend Snippet: Histological and immunohistochemical analyses of in vivo models. ( A ) TUNEL and Ki67 staining of subcutaneous tumor sections from Caco2 and Sw48 xenografts. CTX markedly induced apoptosis (TUNEL) and reduced proliferation (Ki67), whereas NaBu co-treatment attenuated these effects. Scale bars = 100 μm. ( B ) H&E staining of lung sections from pulmonary metastasis models. CTX preserved alveolar architecture and reduced tumor burden, while NaBu co-treatment diminished these protective effects. Scale bars = 500 μm. ( C ) Immunohistochemical staining of subcutaneous tumors showing that NaBu increased p-EGFR and Wip1 expression, consistent with in vitro findings, whereas total EGFR levels were relatively unchanged. Scale bars = 100 μm. The square boxes in the upper-right corners indicate the regions shown at higher magnification in the insets.

Article Snippet: Recombinant human EGFR protein (TargetMol) was fluorescently labeled with Alexa FluorTM 647 NHS Ester (A37573, Invitrogen) according to the manufacturer’s instructions.

Techniques: Immunohistochemical staining, In Vivo, TUNEL Assay, Staining, Expressing, In Vitro

Journal: Drug Design, Development and Therapy

Article Title: Circulating Butyrate Attenuates Cetuximab Efficacy in Colorectal Cancer Through EGFR and AMPK–Wip1 Signaling

doi: 10.2147/DDDT.S574116

Figure Lengend Snippet: Human validation of p-EGFR and Wip1 expression in colorectal cancer. ( A ) Representative immunohistochemical staining of p-EGFR and Wip1 in CRC patient samples stratified by circulating butyrate levels (low vs high). Insets show higher magnification of tumor regions. The square boxes in the upper-right corners indicate the regions shown at higher magnification in the insets. ( B ) Correlation analysis of circulating butyrate concentration with H-scores of p-EGFR (left) and Wip1 (right) in 50 CRC patients, showing significant positive associations (Pearson’s correlation).

Article Snippet: Recombinant human EGFR protein (TargetMol) was fluorescently labeled with Alexa FluorTM 647 NHS Ester (A37573, Invitrogen) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Expressing, Immunohistochemical staining, Staining, Concentration Assay

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Hematoxylin and eosin (H&E) staining of normal duodenum containing Brunner’s glands (nDUO-BG) and duodenal neuroendocrine tumor (DNET). Dashed boxes indicate regions shown at higher magnification. (B) Immunohistochemical staining for synaptophysin (SYP) confirming neuroendocrine differentiation in DNET. (C-D) Immunohistochemical staining for TGFα and EREG in tumor-associated Brunner’s glands (tBG) and DNET. Dashed boxes indicate tumor-gland interfaces. (E-F) Quantification of TGFα and EREG expression by H-score in nDUO-BG, tBG, and DNET. Data are mean ± SEM; ns, not significant; ****P < 0.0001. (G) EGFR immunostaining in nDUO-BG and DNET showing heterogeneous expression across tissues. (H) Menin immunostaining in nDUO-BG and DNET. (I) Representative FFPE DNET specimens showing cytoplasmic or near-absent Menin expression, accompanied by strong TGFα and EREG staining within tumor cells. (J) Quantification of Menin nuclear-to-cytoplasmic (N/C) ratio in nDUO-BG and DNET. Data are mean ± SEM; ****P < 0.0001. Images were taken at 100X, 200X and 400X. Scale bars: 100 μm (low magnification) and 50 μm (high magnification).

Article Snippet: After 24 h, cells were serum-starved for 24 h, followed by treatment with either recombinant human eregulin (EREG) protein (10 nM), a potent ligand for EGFR (R&D systems, #1195-EP) or 10 μM Forskolin (FSK, ThermoFisher, #66575-29-9), activator of adenylyl cyclase and cyclic AMP or and 10nM phorbol 12-myristate 13-acetate (TPA; Sigma-Aldrich, #P8139) for 4-8 h. Cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, #E1980) according to the manufacturer’s instructions.

Techniques: Staining, Immunohistochemical staining, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Multiple sequence alignment of the Menin C-terminal region from the indicated vertebrate species showing strong conservation of a basic residue–rich motif encompassing Ser487. Conserved basic residues and Ser487 are highlighted. (B) Schematic of human Menin illustrating the position of Ser487 within NLS1. The expanded sequence highlights Ser487 and surrounding basic residues; constructs used in this study. (C) Immunoblot analysis of AGS cells expressing FLAG-tagged wild-type Menin or Ser487 mutants (S487A, S487D) following treatment with EREG, FSK, or TPA. Whole-cell lysates were probed with antibodies against phospho-Ser487 Menin, FLAG-Menin, and GAPDH. (D, E) Immunoblot analysis of MKN-45G and KATO III cells expressing wild-type Menin following stimulation with EREG, FSK, or TPA. Blots were probed for phospho-Ser487 Menin, FLAG-Menin, and β-tubulin. (F, H) Quantification of phospho-Ser487 Menin in AGS, KATO III and MKN-45G cells. (I) Immunoblot analysis of AGS cells examining activation of cAMP and EGFR downstream kinases under the indicated conditions. (J) Densitometric quantification of signalling outputs shown in (I), expressed as fold change relative to vector control. (K) Time-course of Ser487 phosphorylation in AGS cells stimulated with TPA in the presence of kinase inhibitors; MEK inhibitor (U0126), AKT inhibitor (MK-2206), PKC inhibitor (Gö6983), or combined MEK+AKT inhibition. (L) Quantification of Ser487 phosphorylation kinetics following TPA stimulation with the indicated inhibitors. (M) Area-under-the-curve (AUC) analysis of phosphorylation in (L). Data are presented as mean ± SEM; individual data points represent independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant).

Article Snippet: After 24 h, cells were serum-starved for 24 h, followed by treatment with either recombinant human eregulin (EREG) protein (10 nM), a potent ligand for EGFR (R&D systems, #1195-EP) or 10 μM Forskolin (FSK, ThermoFisher, #66575-29-9), activator of adenylyl cyclase and cyclic AMP or and 10nM phorbol 12-myristate 13-acetate (TPA; Sigma-Aldrich, #P8139) for 4-8 h. Cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, #E1980) according to the manufacturer’s instructions.

Techniques: Sequencing, Residue, Construct, Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Phospho-proteomics, Inhibition, Comparison