Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: Characterization of hPD1(25-167)-His and hPD1(25-167)-3S-IG

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–c Characterization of the assay reference standard-recombinant hPD1(25-167)-His protein. a SDS-PAGE analysis of the purified hPD1(25-167)-His protein expressed in HEK293 cells. Lane 1: molecular weight marker, lane 2: blank, lane 3: 1 μg of the purified hPD1(25-167)-His protein. The gel was stained with Simply Blue SafeStain. b Concentration series of hPDL-1 (0.125–2 μM) binding to captured hPD1(25-167)-His protein. c Equilibrium analysis of hPDL-1 binding to hPD1(25-167)-His yields a K D of 2.8 μM (average value over multiple surfaces)

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Recombinant, SDS Page, Purification, Molecular Weight, Marker, Staining, Concentration Assay, Binding Assay

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: Binding Affinity of Anti-PD-1 Antibody MIH4 for Recombinant Human sPD-1 Proteins

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Binding Assay, Recombinant

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Detection of endogenous sPD-1 protein and spike recovery in sera from normal and cancer individuals. a Levels of sPD-1 in serum samples from normal and cancer individuals: normal, n = 15; melanoma, n = 15; renal cell carcinoma, n = 11; squamous lung cancer, n = 7; non-squamous lung cancer, n = 8; multiple myeloma, n = 10; Hodgkin’s lymphoma, n = 7. b Spike and recovery study: 500 pg/mL hPD1(25-167)-His protein was spiked into serum samples from normal and cancer individuals: normal, n = 10; melanoma n = 8; renal cell carcinoma, n = 11; squamous lung cancer, n = 7; non-squamous lung cancer, n = 8; multiple myeloma, n = 10; Hodgkin’s lymphoma, n = 7. % Spike recovery = 100*(sample value post-spike − sample value pre-spike)/spiked value. Results are shown with box and whiskers graphs. The box included data within 5 to 95 percentiles with the median line in the middle and extended values in the whiskers. The dotted lines depict the nominal spiked value ± 25%

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Dilution linearity studies in melanoma and spiked normal sera. a Three melanoma serum samples (M1, M2, M3) were each diluted 1.5–8-fold with assay buffer; recombinant hPD1(25-167)-His protein standard in a SeraSub solution was diluted in same concentration range. b Three normal human serum samples (N1, N2, N3) were spiked with 5000 pg/mL of human hPD1(25-167)-His and were each diluted 2–256-fold with assay buffer. The back-calculated sPD-1 concentrations (observed concentrations times the dilution factor) were plotted against dilution factors. The dotted lines depict 75–125% range of the nominal spiked value

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Recombinant, Concentration Assay

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Effect of PDL-1, PDL-2, and nivolumab on QC sample performance. a sPD-1 in a melanoma serum sample measured in the absence or presence of 20 nM (1 μg/mL) of human PDL-1 or PDL-2 proteins. b QC performance from analytical runs in the absence or presence of 685 nM (100 μg/mL) nivolumab. Results are shown as mean and SD of each group (n = 2–4)

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP forms a ternary complex with regulatory RIIα subunits of PKA and GSK3β and facilitates PKA phosphorylation of GSK3β. A, left panel, wells of microtiter plates were coated with His-GSKIP (500 nm) and incubated with GSK3β (0.25–64 nm) in blocking buffer or with blocking buffer without GSK3β. GSK3β was detected with anti-GSK3β and secondary HRP-conjugated antibodies and an HRP-catalyzed reaction with a chromogenic substrate solution. Right panel, wells of microtiter plates were coated with RIIα (50 nm) in blocking buffer or with blocking buffer in the absence of RIIα. GSKIP (250 nm) in combination with increasing amounts of GSK3β (0.25–64 nm) was added. To quantify unspecific signals, the GSKIP-GSK3β-combination was incubated in wells blocked in the absence of RIIα (▵). Direct binding of GSK3β to RIIα was assessed by incubation of RIIα-coated wells with GSK3β (0.25–64 nm; ○). Detection was carried out as above. B, left panel, CFP or CFP-GSKIP was transiently expressed in HEK293 cells. The cells were left untreated, incubated with the direct activator of adenylyl cyclases, forskolin (20 μm, 30 min), or pretreated with the PKA inhibitor H89 (30 μm, 30 min prior to FSK treatment). Cells were lysed, and CFP-GSKIP, phosphorylated (p-GSK3β), and total GSK3β were detected with specific antibodies by Western blotting (WB). Right panel, signals obtained for p-GSK3β and GSK3β by Western blotting were analyzed densitometrically. The ratio of p-GSK3β to GSK3β was calculated (n = >5 independent experiments for each experimental condition, mean ± S.E.). C, left panel, HEK293 cells were treated with FSK or a combination of FSK and H89 as in B or with stearate-coupled GSKIPtide, which displaces GSK3β from GSKIP (20), or the inactive control peptide GSKIPtide-L130P (100 μm, 30 min). Cells were lysed and phosphorylated GSK3β (p-GSK3β) and GAPDH (loading control) were detected by Western blotting. Right panel, signals obtained for p-GSK3β and GAPDH by Western blotting were analyzed densitometrically. The ratio of p-GSK3β to GAPDH was calculated (n = 7 independent experiments for each experimental condition, mean ± S.E.). B and C, significantly different from untreated control: #, p < 0.05; ##, p < 0.01; ###, p < 0.001. Significant differences between groups connected by lines: *, p < 0.05; **, p < 0.01). n.s., not significant.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: Phospho-proteomics, Incubation, Blocking Assay, Binding Assay, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP functions as an AKAP in vitro. A, GSKIP and the DD domain of human regulatory RIIα subunits of PKA were generated and NMR measurements performed. 1H-15N heteronuclear single quantum coherence spectra of the GSKIP domain in the absence (red) or presence of DD (black) at 1:0.5 ratio are shown. Disappearance of the GSKIP resonances is caused because of chemical exchange broadening and/or increased transverse relaxation rate due to high molecular weight of the complex. Lower panel, Protein Data Bank structure of GSKIP (Protein Data Bank code 1sgo) The RII-binding domain (black) encompasses the amphipathic α1-helix of GSKIP. The conserved hydrophobic amino acid residues Ala-37, Val-40, Val-41, Val-44, Leu-45, and Val-48 (see also Fig. 1A) are depicted as ball-and-stick models. The contact interface for RII binding is hidden by a β-sheet. B, surface plasmon resonance measurements to determine the association and dissociation rate constants (see Table 2) for the binding of GSKIP to PKA regulatory RII subunits. Human RIIα and RIIβ subunits (upper and lower panel, respectively) were captured on 8-AHA-cAMP sensor chips, and His-GSKIP was injected into the Biacore instrument in the indicated concentrations. The plots show representative experiments. Each experiment was repeated at least three times using different protein preparations and different immobilization rates of the regulatory subunits. RU, resonance units. C, immunoprecipitated (IP) GSKIP binds RII subunits. Cyan fluorescent protein (CFP; lane 1) and the indicated fusions of human full-length GSKIP with CFP (lane 2) and GSKIP-V41P/L45P with CFP (lane 4) were transiently expressed in HEK293 cells. CFP-GSKIP-V41P/L45P contains prolines in the RII-binding domain disrupting the α-helical structure and thus preventing the interaction with RII. The cells were lysed, and GSKIP was immunoprecipitated with anti-GSKIP antibodies, or the precipitation was carried out with the corresponding preimmune serum. Precipitated proteins were probed for RII binding by RII overlay assay in the absence (control) or in the presence of the PKA anchoring disruptor peptide, AKAP18δ-L314E (10 μm). Signals were detected by autoradiography. Lane 3, molecular weight standard. Lower panel, Western blot (WB) with anti-green fluorescent protein antibody (also detects CFP (26)) for the detection of CFP, CFP-GSKIP, or CFP-GSKIP-V41P/L45P in HEK293 cells transiently expressing the proteins. Proteins were detected in precipitates obtained with rabbit anti-GSKIP antibody (IP, left panel) or obtained with preimmune serum (middle panel) or in cell lysates.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: In Vitro, Generated, High Molecular Weight, Binding Assay, SPR Assay, Injection, Immunoprecipitation, Overlay Assay, Control, Autoradiography, Molecular Weight, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP is widely expressed and functions as an AKAP in vivo. A, lysates from the indicated rat organs were subjected to Western blot (WB) analysis with an anti-GSKIP antibody and, as a loading control, anti-GAPDH antibody (167 μg of total protein per lane). B, cAMP-agarose pulldowns were obtained from the indicated rat tissue lysates (3 mg of total protein in each sample) in the absence (− cAMP) or presence (+ cAMP; 50 μm) of cAMP. RIIα subunits of PKA and GSKIP were detected with specific antibodies by Western blotting. skel., skeletal. C, indicated subcellular fractions were obtained from SH-SY5Y cells. 10 μg of total protein from each fraction was analyzed for the presence of the indicated proteins by Western blotting. D, GSKIP and RIIα co-localize in HEK293 cells. HEK293 cells were transiently transfected to express YFP-RIIα and fusions of CFP with wild type GSKIP (CFP-GSKIP WT), GSKIP-V41P/L45P, or as a plasma membrane marker AKAP18α.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: In Vivo, Western Blot, Control, Transfection, Clinical Proteomics, Membrane, Marker

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP contains the evolutionarily conserved DUF727, which binds PKA and GSK3β. A, RII-binding domain of GSKIP is located within DUF727. Shown are sequence alignments of the RII-binding domain of GSKIP with orthologues from the indicated vertebrate and invertebrate species (upper and lower, panels, respectively). Swiss-Prot protein identification codes (Prot.-ID) and positions of the RII-binding domains within the cognate proteins are indicated (for Canis familiaris, the NCBI protein accession is given as no Swiss-Prot entry was available). Amino acid residues in conserved positions of RII-binding domains are shaded in gray (see also Fig. 1A). Peptides depicted in the alignments were spot-synthesized and assayed for RII binding in the absence (control) or the presence of the PKA anchoring disruptor peptide AKAP18δ-L314E (10 μm). Signals were detected by autoradiography. Numbers of the spots correspond to the respective numbers in the alignments. Shown are representative membranes from three independent experiments. B, GSK3β binding is conserved in GSKIP orthologues. Left panel, alignment of all 51 DUF727 proteins with the C terminus of human GSKIP (amino acids 115–139) and human axin-1 and -2. Hydrophobic residues required for GSK3β interaction, which are identical (dark gray) or similar (bright gray) to human axin-1, are highlighted. Right panel, peptide spots of corresponding sequences in the alignment were spot-synthesized and incubated with GST-GSK3β or GST as a negative control. Peptides marked with a P contain an Leu → Pro (L→P) mutation in the position corresponding to amino acid 130 in human GSKIP (asterisk in left panel). Protein binding was detected with an anti-GST antibody. Both axin peptides and 40 of 50 DUF727 peptides bound GST-GSK3β. This result is representative for three independent experiments.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: Binding Assay, Sequencing, Synthesized, Control, Autoradiography, Incubation, Negative Control, Mutagenesis, Protein Binding

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 1. Prediction of Tpr2 structural domains. (A) Domain prediction revealed seven tetratricopeptide motifs in human Tpr2 (motifs 1–7). Two sets of three motifs were predicted to form dicarboxylate clamp domains (T1 and T2). A DnaJ homology domain (J) near the C-terminus was also identified. The boundaries of the predicted domains are indicated as amino acid residue numbers. Introduced point mutations (dT1, dT2 and dJ) are listed with the respective amino acid exchange. (B) Sequences of the predicted T1 and T2 domains in Tpr2 were aligned against the Hsp70- and Hsp90-binding dicarboxylate clamp domains of Hop, TPR1 and TPR2A, respectively. Conserved residues that participate in the formation of the dicarboxylate clamp are underlined in boldface. Residues in boldface alone determine the specificity of chaperone binding. The arginine residues mutated to alanine in the dT1 and dT2 point mutants are marked with an asterisk. (C) The sequence of the predicted J domain in Tpr2 was aligned against the J domains of Hsp40 and Hdj-2. The functional HPD motif is underlined in boldface. Conserved residues are marked in boldface. The histidine residue mutated to alanine in the dJ point mutant is marked with an asterisk.

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Residue, Binding Assay, Sequencing, Functional Assay, Mutagenesis

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 2. Perturbation of Tpr2 expression levels reduce glucocorticoid receptor (GR) activation in vivo. Cells were transfected with a plasmid encoding a luciferase reporter gene downstream of a GR response element (GRE) and a control plasmid encoding β-galactosidase. Cells were treated for 24 h with 1 µM dexamethasone where indicated and harvested. Cell lysates were tested for luciferase activity and normalized against β-galactosidase activity. Samples were immunoblotted with antibodies against Tpr2, Hop or GR. In all figures, error bars show standard deviations from the mean of at least three independent experiments. (A) Empty vector (lanes 1–2) or vectors encoding myc-tagged Tpr2 (lanes 3–4), myc-tagged Hop (lanes 5–6) or both Tpr2 and Hop (lanes 7–8) were co-transfected into N2A cells together with the reporter and control plasmids. Top, immunoblot with antibodies against Tpr2 or Hop; transfected overexpressed myc-tagged proteins (o) are visible as bands above endogenous species (e). Bottom, GR-activated normalized luciferase expression in cells under conditions indicated. Columns correspond to the above immunoblot. (B) A double-stranded siRNA oligomer against the Tpr2 RNA was co-transfected into HeLa cells with the reporter and control plasmids (lanes 3–4). Control experiments included either an empty vector (lanes 1–2) or double-stranded RNA oligomers with a scrambled sequence (scRNA, lanes 5–6) or mutated sequence (mutRNA, lanes 7–8). Top, immunoblot against endogenous Tpr2 (e). Bottom, normalized GR-activated luciferase expression under indicated conditions. Columns correspond to the above immunoblot. (C) The indicated total cell lysates were resolved on SDS–PAGE and immunoblotted for GR, and for actin as a loading control. (D) Immunofluorescence of cells treated with siRNA against Tpr2 or control cells. Nuclei are stained with DAPI. Scale bar represents 100 µm. (E) Empty vector or vectors encoding Tpr2, GRΔLBD or both vectors were co-transfected with the reporter and control plasmids. Normalized GR-activated luciferase expression under indicated conditions are plotted.

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Expressing, Activation Assay, In Vivo, Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay, Western Blot, Sequencing, SDS Page, Immunofluorescence, Staining

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 3. Hsp90 and Hsp70 are the two major interaction partners of Tpr2. (A) Purified His-tagged wild-type (WT) Tpr2, Tpr2 point mutated in the TPR clamp domains (dT12) or J-domain (dJ), or with the combination of point mutations (dT12J) were tested for binding to reticulocyte lysate (RL) proteins. The indicated proteins at 10 µM concentration were incubated with RL, in parallel with a control reaction with no added protein (Ni-NTA). Complexes were recovered with Ni-NTA agarose and bound proteins eluted with 500 mM NaCl (top). Tpr2 proteins were re-eluted with SDS sample buffer containing 25 mM EDTA (bottom). Samples were resolved by SDS–PAGE and visualized by Coomassie blue staining. The two major bands eluting from WT Tpr2 were identified by immunoblotting as Hsp90 and Hsp70. The position of molecular weight standards is marked on the right. (B) 10 µM Tpr2 together with 50 µM of the C-terminal fragments of Hsp90 (90C, lane 3) or Hsp70 (70C, lane 4) were present during the binding reaction. After recovery with Ni-NTA agarose and elution with 500 mM NaCl, eluted proteins were resolved on SDS–PAGE and detected by immunoblotting against Hsp90 and Hsp70.

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Purification, Binding Assay, Concentration Assay, Incubation, Control, SDS Page, Staining, Western Blot, Molecular Weight

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 4. Quantitative analysis of the Tpr2–chaperone interactions. 12mer peptides containing the C-terminal sequence of either Hsp70 or Hsp90 (70C-12 and 90C-12 respectively) were covalently coupled to a Biacore chip. Various concentrations of Tpr2 and its mutants were injected and the association and dissociation monitored by the surface plasmon resonance (SPR) signal. (A) Binding kinetics of Tpr2 in the concentration range of 0.1–30 µM were monitored (right panel) with immobilized 90C-12 or 70C-12. The relative response units during the equilibrium phase of binding to 90C-12 or 70C-12 were plotted against Tpr2 concentrations (left panel). (B) Binding efficiency to 70C-12 or 90C-12 of wild-type (WT) or indicated point mutants of Tpr2 at a constant protein concentration of 1 µM was tested. The relative response units during equilibrium binding were plotted. (C and D) Increasing concentrations (0.1–100 µM) of 70C-12 or 90C-12 in solution were used to compete for binding of Tpr2 to immobilized 70C-12 (C) or 90C-12 (D). A control peptide terminating in SKL, which is recognized by the TPR domain of Pex5p, but not by Hop, was also tested. Binding kinetics were monitored (right panels) and Tpr2 binding as a percentage of the control without soluble peptides was plotted against soluble peptide concentration (left panels).

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Sequencing, Injection, SPR Assay, Binding Assay, Concentration Assay, Protein Concentration, Control

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Thermodynamic binding constants ( K D ) of wild-type and mutant Tpr2 to the Hsp70 and Hsp90 C-termini

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Binding Assay, Mutagenesis

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 5. The Tpr2 J domain regulates Hsp70. (A) 1 µM Hsc70/Hsp70, 2 µM Hsp40 and 2 µM wild-type or mutant Tpr2 in the indicated combinations were incubated at 30°C in the presence of [α-32P]ATP and 0.1 mM ATP. Aliquots of each reaction were stopped at different time points with 25 mM EDTA, resolved by thin layer chromatography and evaluated by PhosphorImager scanning. Steady-state ATPase rates were calculated from the linear range of the reactions. (B) Purified, partially-folded GR ligand binding domain (LBD) was bound to Ni-NTA agarose and beads were incubated for 10 min at room temperature with 5 µM Hsc70/Hsp70 and either no added protein (buffer), 5 µM wild-type (WT) or point mutated Tpr2, or Hsp40. Beads were recovered and bound proteins eluted with SDS sample buffer. Samples were resolved by SDS–PAGE and visualized by Coomassie blue staining. (C) Guanidine-denatured luciferase was diluted 100-fold into reactions containing 3% RL and ATP, supplemented with buffer or 1 µM Hsc70/Hsp70 with 2 µM Hsp40 or Tpr2 where indicated. Luciferase refolding at 30°C was monitored over time and plotted as a percentage of the activity of native luciferase.

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Mutagenesis, Incubation, Thin Layer Chromatography, Purification, Ligand Binding Assay, SDS Page, Staining, Luciferase, Activity Assay

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 6. Tpr2 dissociates Hsp90 but not Hsp70 from substrate polypeptide complexes. Partially-folded myc-His-tagged ligand binding domain (LBD) or guanidine-denatured myc-tagged luciferase were pre-bound to anti-myc antibodies covalently coupled to protein G–Sepharose. Radiolabelled wild-type Hsp90, or the Hsp90 D93N point mutant unable to bind ATP, or Hsp70 was generated by in vitro translation. The translation reactions were added to reticulocyte lysate (RL) containing ATP and chaperone–substrate complexes were formed for 10 min at 25°C and then immune-isolated. The dissociation of radiolabelled chaperones was assayed under different conditions. After 10 min of dissociation at 25°C, beads and supernatants were separated and proteins in the supernatants precipitated. Released and bound proteins were resolved by SDS–PAGE and quantified by PhosphorImager analysis. The fraction of radiolabelled chaperones released from the complexes was plotted as a percentage of the total. (A) Dissociation of wild-type Hsp90 from LBD was measured in the presence of buffer alone, or 5 µM p23 or Tpr2, without or with 2 mM ATP. (B) Dissociation of Hsp90 D93N from LBD was measured in the presence of buffer alone, or 5 µM p23 or Tpr2, without or with 2 mM ATP. (C) Dissociation of Hsp90 D93N from luciferase was measured in the presence of buffer alone, or 5 µM p23 or Tpr2, without or with 2 mM ATP. (D) Dissociation of Hsp90 D93N from LBD was measured in the presence of buffer alone, or 5 µM wild-type or point mutated Tpr2. (E) Dissociation of Hsp70 was measured in the presence of buffer alone, or 5 µM Bag-1, or 5 µM wild-type or point mutated Tpr2.

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Ligand Binding Assay, Luciferase, Mutagenesis, Generated, In Vitro, Isolation, SDS Page

Journal:

Article Title: Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

doi: 10.1093/emboj/cdg362

Figure Lengend Snippet: Fig. 7. The different domains of Tpr2 cooperate to regulate the Hsp70/Hsp90 machinery. (A) Glucocorticoid receptor (GR) activation in N2A cells with or without dexamethasone was determined as in Figure 2A. Empty vector, vectors encoding wild-type or mutant Tpr2 were co-transfected with the reporter and control plasmids. Top, immunoblot against overexpressed (o) and endogenous (e) Tpr2 forms. Bottom, normalized GR-activated luciferase expression under indicated conditions. (B) Full-length GR was added to reticulocyte lysate (RL) supplemented with various purified proteins and 2 mM ATP. Reactions were incubated at 42°C for 5 min to unfold the GR, then at 30°C for 15 min to allow chaperone-mediated refolding of the GR. [3H]Dexamethasone was allowed to bind the GR, unbound hormone was removed by fast gel filtration and bound hormone quantified by scintillation counting. Hormone binding by GR refolded in RL with no additions was set to 100%. As a negative control, RL was treated with 40 µM GA to inhibit Hsp90, or with an equivalent volume of DMSO as a solvent control. Other reactions were supplemented with 2 µM wild-type, mutant Tpr2 or Hop. (C) Hormone binding by GR after refolding in RL supplemented with increasing concentrations of wild-type Tpr2 was measured and plotted against the concentrations of Tpr2 added.

Article Snippet: Cell culture DNA sequences encoding Tpr2 or human Hop were inserted into pcDNA3.1 (Invitrogen) in-frame with the C-terminal myc-tag.

Techniques: Activation Assay, Plasmid Preparation, Mutagenesis, Transfection, Control, Western Blot, Luciferase, Expressing, Purification, Incubation, Filtration, Binding Assay, Negative Control, Solvent

Journal: Communications Biology

Article Title: Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration

doi: 10.1038/s42003-022-04305-9

Figure Lengend Snippet: A Relative binding of VEGF to 52 different synthetic glycosaminoglycans measured on a microarray. B An illustration of the microarray. C Relative binding of HGF in the microarray to show the difference in binding pattern depending on the glycosaminoglycan. For A and C data is shown in a box and whisker (as Tukey) of 36 binding spots for each synthetic glycosaminoglycan. D Six synthetic glycosaminoglycans with different binding patterns were chosen for further analyses using surface plasmon resonance (SPR) to determine their binding in a quantitative way compared to the semi quantitative analysis that the microarray allows. E Heatmap showing the binding of six selected synthetic glycosaminoglycans on the x -axis and the tested growth factors on the y -axis. The glycosaminoglycans are ordered in increasing degree of sulfation, GAG nr 3, 43, 34, 10, 26, and 19. The color shows the k off value, where red illustrates a slow release while a blue color a fast release. The size of the circle illustrates the equilibrium dissociation constant, K d , where a larger circle is for higher binding while a smaller circle is for lower binding. There are also two points with no data indicated in gray and two points that did not show any release at all, indicated in green. For convenience, we have highlighted the growth factor FGF2 (gray line) and GAG nr 19 used in later experiments (orange highlight). Factors tested that did not show any interactions in the SPR analysis were: IL- 6, IL-11, TGF beta 2, TGF beta 3, CCL2, CCL3, CCL4, CCL23, Wnt2, G-CSF, NOV, EGF, IGF1, GLP, SCF, CXCL2, CXCL7.

Article Snippet: The slides were rinsed in PBST with 1% BSA for 3 min and then in deionized water for 3 min. Antibodies towards the protein were dissolved in PBST 10% BSA according to the manufacturer’s recommended concentration, added to the slide and incubated in a humified chamber for 60 min (HGF Ab, MAB694, 0.4 μg/ml, Mouse anti Human, RnD systems) (VEGF Ab, MAB293, 0.4 μg/ml, Mouse anti Human, RnD systems) (BMP4 Ab, MAB7571, 0.4 μg/ml, Mouse anti Human, RnD systems) (CXCL12 Ab, MAB350, 0.4 μg/ml, Mouse ant Human, RnD Systems) (IL-6 Ab, MAB2063, 0.4 μg/ml, Mouse ant Human, RnD systems) (FGF2 Ab, ab92337, 0.5 μg/ml, Rabbit anti Human, Abcam) (PDGF-AA Ab, MAB221, 0.4 μg/ml, Mouse anti Human, RnD systems) (KGF Ab, MAB251, 0.4 μg/ml, Mouse anti Human, RnD systems) (TGF beta 1 Ab, MAB240, 0.4 μg/ml, Mouse anti Human, RnD systems).

Techniques: Binding Assay, Microarray, Whisker Assay, SPR Assay

Journal: Communications Biology

Article Title: Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration

doi: 10.1038/s42003-022-04305-9

Figure Lengend Snippet: Growth factors tested and the molecular weight according to the manufacturer, the theoretical pI, the highest concentration tested for the SPR experiment as well as the lowest concentration tested.

Article Snippet: The slides were rinsed in PBST with 1% BSA for 3 min and then in deionized water for 3 min. Antibodies towards the protein were dissolved in PBST 10% BSA according to the manufacturer’s recommended concentration, added to the slide and incubated in a humified chamber for 60 min (HGF Ab, MAB694, 0.4 μg/ml, Mouse anti Human, RnD systems) (VEGF Ab, MAB293, 0.4 μg/ml, Mouse anti Human, RnD systems) (BMP4 Ab, MAB7571, 0.4 μg/ml, Mouse anti Human, RnD systems) (CXCL12 Ab, MAB350, 0.4 μg/ml, Mouse ant Human, RnD Systems) (IL-6 Ab, MAB2063, 0.4 μg/ml, Mouse ant Human, RnD systems) (FGF2 Ab, ab92337, 0.5 μg/ml, Rabbit anti Human, Abcam) (PDGF-AA Ab, MAB221, 0.4 μg/ml, Mouse anti Human, RnD systems) (KGF Ab, MAB251, 0.4 μg/ml, Mouse anti Human, RnD systems) (TGF beta 1 Ab, MAB240, 0.4 μg/ml, Mouse anti Human, RnD systems).

Techniques: Molecular Weight, Concentration Assay

Journal: PLoS Genetics

Article Title: GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

doi: 10.1371/journal.pgen.1004305

Figure Lengend Snippet: (A) Western blotting from adult testis extracts. Polyclonal antibodies raised against GOLPH3 protein, recognized a band of the predicted molecular weight. The band is reduced in testis extracts from sau z2217 / Df(2L)Exel7010 and GOLPH3RNAi males. α-Tubulin was used as a loading control. (B) Colocalization of GOLPH3 (anti-GOLPH3, red) with the Golgi protein Cog7-GFP (green) in interphase spermatocytes. (C) In dividing spermatocytes GOLPH3 protein (red) was enriched in Golgi derived vesicles and accumulated at the cleavage furrow (arrows) during telophase (green) Tubulin; (blue) DNA. Scale Bar; 10 µm.

Article Snippet: Preparations fixed to visualize Clc-GFP together with tubulin, were first incubated with monoclonal anti-α–Tubulin (see above) followed by incubation with GFP-Booster (ChromoTek) diluited 1∶100 in PBS containing rhodamine-conjugated anti-mouse IgG.

Techniques: Western Blot, Molecular Weight, Control, Derivative Assay

Journal: PLoS Genetics

Article Title: GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

doi: 10.1371/journal.pgen.1004305

Figure Lengend Snippet: (A) Frequencies of spermatids (percentage) containing 1, 2, 3, 4 or more than 4 nuclei (nu) per Nk in testes from males of the indicated genotypes. (n); total number of spermatids counted per each genotype. Scale Bar; 10 µm. (B) Live spermatocytes of the indicated genotypes, expressing either GFP-GOLPH3 or a mutant version of GFP-GOLPH3 carrying K167A/R170L substitutions (GFP-GOLPH3 K167A ) were imaged at the same exposure time. GFP-GOLPH3 failed to localize to Golgi stacks (fluorescent round bodies indicated by arrows) in fwd 3 /Df(3L)7C [fwd 3 /Df] mutant spermatocytes. GFP-GOLPH3 K167A failed to localize at the Golgi membranes in both wild type and sau z2217 /Df(2L)Exel7010 ( sau z2217 /Df ) mutant spermatocytes. (C) Phase-contrast and corresponding fluorescence images of live telophase spermatocytes expressing either GFP-GOLPH3 (GOLPH3) or GFP-GOLPH3 carrying K167A/R170L substitutions (GOLPH3K 167A ). Arrows indicate cleavage furrows. Scale Bar; 10 µm. (D) PIP strips (Echelon) were incubated with the indicated GST-tagged protein or GST, followed by anti-GST antibody for detection of lipid binding. PI(3)P ctrl and PI(4)P ctrl indicate GST-tagged control proteins that bind to either PI(3)P or PI(4)P. (E) Immunoblotting analysis of wild type testes expressing either GFP-GOLPH3 (wild type), GFP-GOLPH3 E273K (E273K) or GFP-GOLPH3 K167A/R170L (K167A). Giotto (Gio) was used as a loading control.

Article Snippet: Preparations fixed to visualize Clc-GFP together with tubulin, were first incubated with monoclonal anti-α–Tubulin (see above) followed by incubation with GFP-Booster (ChromoTek) diluited 1∶100 in PBS containing rhodamine-conjugated anti-mouse IgG.

Techniques: Expressing, Mutagenesis, Fluorescence, Incubation, Binding Assay, Control, Western Blot

Journal: PLoS Genetics

Article Title: GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

doi: 10.1371/journal.pgen.1004305

Figure Lengend Snippet: (A,C) Live spermatocytes expressing either RFP-PH-FAPP (RFP-FAPP) or PLCδ-PH-GFP were imaged at the same exposure time during telophase. Note that the distribution of both RFP-PH-FAPP and PLCδ-PH-GFP is affected in GOLPH3 mutant spermatocytes indicating a defective PI(4)P and PI(4,5)P2 subcellular localization and/or synthesis. Arrows indicate accumulation of RFP-PH-FAPP (A) and PLCδ-PH-GFP (C) at the cleavage furrows of wild type cells. (B) Fixed telophase spermatocyes expressing RFP-PH-FAPP (RFP-FAPP) stained for RFP (red), tubulin (green) and DNA (blue). Arrows indicate the cell midzone. Arrowheads indicate the enrichment of RFP signals at the poles of the wild type cell. Scale Bar; 10 µm.

Article Snippet: Preparations fixed to visualize Clc-GFP together with tubulin, were first incubated with monoclonal anti-α–Tubulin (see above) followed by incubation with GFP-Booster (ChromoTek) diluited 1∶100 in PBS containing rhodamine-conjugated anti-mouse IgG.

Techniques: Expressing, Mutagenesis, Staining

Journal: PLoS Genetics

Article Title: GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

doi: 10.1371/journal.pgen.1004305

Figure Lengend Snippet: (A) Telophase spermatocytes from wild type and sau z2217 /Df(2L)Exel7010 (GOLPH3) stained for Tubulin (green), Rab11 (red) and DNA (blue). Arrows indicate accumulation of Rab11 at the cleavage furrow of wild type spermatocyte. Scale Bar; 10 µm. (B) GOLPH3 protein coprecipitated with Rab11 and Rab5 in testis extracts. Protein extracts from testes expressing either GFP-Rab5 or GFP-Rab11 were immunoprecipitated with GFP-trap beads (α-GFP) and blotted for either GFP or GOLPH3. Control binding beads (ctrl) were used in control experiments. Input is 4% of lysates. Molecular masses are indicated in kilodaltons. (C) Bacterially expressed GST-GOLPH3 was purified by gluthatione-sepharose beads and incubated with testis lysates expressing GFP-Rab11. GST bound to gluthatione-sepharose beads was used as a negative control. GST-GOLPH3 precipitated GFP-Rab11 from testis protein extracts. Ponceau staining (Ponceau) is shown as loading control. Input is 4% of lysates. Molecular masses are indicated in kilodaltons. (D) Interaction of GOLPH3 immobilized on a CM5 sensorchip with Rab11, measured via Surface Plasmon Resonance experiments. Sensorgrams were obtained using GOLPH3 as ligand and Rab11 as analyte. Rab11 concentrations were as follows: 50 nM (orange), 200 nM (red), 400 nM (magenta), 1.0 µM (green), 2.0 µM (cyan), 4.0 µM (blue), 10.0 µM (black). The interaction of immobilized GOLPH3 ligand with the analyte was detected through mass concentration-dependent changes in the refractive index on the sensor chip surface, expressed as resonance units (RU). The increase in RU relative to baseline (0–180 s) indicates complex formation, whereas the decrease in RU represents dissociation of Rab11 from immobilized GOLPH3 upon injection of buffer. K D = 180 nM indicates that a high affinity complex is formed; the dissociation kinetics are very slow (k d about 1×10 −4 s −1 ).

Article Snippet: Preparations fixed to visualize Clc-GFP together with tubulin, were first incubated with monoclonal anti-α–Tubulin (see above) followed by incubation with GFP-Booster (ChromoTek) diluited 1∶100 in PBS containing rhodamine-conjugated anti-mouse IgG.

Techniques: Staining, Expressing, Immunoprecipitation, Control, Binding Assay, Purification, Incubation, Negative Control, SPR Assay, Concentration Assay, Refractive Index, Injection

Journal: PLoS Genetics

Article Title: GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

doi: 10.1371/journal.pgen.1004305

Figure Lengend Snippet: (A,B) Co-IP of GOLPH3 with Zipper and Septins. (A) Equal fractions of protein extracts from testes expressing GFP-GOLPH3 were immunoprecipitated with either mouse anti-GFP (α-GFP) or non-specific mouse IgG (ctrl) and blotted for either Zipper or GFP. (B) Protein extracts from wild type testes were immunoprecipitated with antibodies against Drosophila GOLPH3 (α-GOLPH3) and blotted for either Sep2, Sep1 or GOLPH3. Preimmune serum was used as control (ctrl). (C) Bacterially expressed GST-GOLPH3 was purified by gluthatione-sepharose beads and incubated with testis lysate. GST bound to gluthatione-sepharose beads was used as a negative control. GST-GOLPH3 precipitated Zipper, Pav and Sep1 from testis protein extracts. Ponceau staining (Ponceau) is shown as a loading control. In all the experiments input is 4% of lysates. Molecular masses are indicated in kilodaltons.

Article Snippet: Preparations fixed to visualize Clc-GFP together with tubulin, were first incubated with monoclonal anti-α–Tubulin (see above) followed by incubation with GFP-Booster (ChromoTek) diluited 1∶100 in PBS containing rhodamine-conjugated anti-mouse IgG.

Techniques: Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Control, Purification, Incubation, Negative Control, Staining

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Surface plasmon resonance analysis of sclerostin/GAG interaction, reported as resonance units (RU). ( A ) Relative binding levels of labeled and unlabeled sulfated or native hyaluronan and corresponding sensorgrams ( B ). GAGs were injected at 50 and 500 µM related to the molecular weight of their disaccharide units (D.U.).

Article Snippet: Recombinant human Sclerostin (R&D Systems, Minneapolis, MN, USA) was immobilized onto Series S Sensor Chips CM5 via amine coupling at 25 °C as described by GE Healthcare, resulting in immobilization values of about 2150 RU.

Techniques: SPR Assay, Binding Assay, Labeling, Injection, Molecular Weight

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Sclerostin binding to variously decorated material films. ( A – C ) Differently modified surfaces were incubated with 8 ng/mL sclerostin. Binding was quantified by the amount of unbound sclerostin in the supernatant after 24 h ( A , B ). ( C ) Cumulative sclerostin release from a saturated and preincubated (40 ng/mL) sHA3 surface. Sclerostin was presented in either 0.1% BSA and 0.2% Tween 80 in PBS ( A ) or DMEM with 10% serum ( B , C ). Film surfaces: sHA3: covalent ED900 and sHA3-coating; ED900: ED900 modified film preincubated with soluble sHA3, Ac. ED900: Acetylated ED900 acetylated to neutralize positive charge; T134LA6: nonmodified film. All values represent the mean ± SD of n ≥ 4. *** p < 0.001 denotes significant difference vs. the respective surface modification.

Article Snippet: Recombinant human Sclerostin (R&D Systems, Minneapolis, MN, USA) was immobilized onto Series S Sensor Chips CM5 via amine coupling at 25 °C as described by GE Healthcare, resulting in immobilization values of about 2150 RU.

Techniques: Binding Assay, Modification, Incubation

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Sclerostin and DKK1 are the preferred binding partner of sHA3 functionalized films. Differently modified films were incubated with the same molar amounts of Sclerostin, Wnt3a, or both proteins combined. Unbound ( A ) Sclerostin, ( B ) Wnt3a, and ( C ) DKK1 was quantified after 24 h from the supernatant via ELISA. Recovery normalized to controls without any films (blank well plates). Film surfaces: ED900: ED900 incubated with soluble sHA3; sHA3: ED900 covalently coated with sHA3. All values represent the mean ± SD of n ≥ 4. *** p < 0.001 denotes significant difference vs. the respective surface modification.

Article Snippet: Recombinant human Sclerostin (R&D Systems, Minneapolis, MN, USA) was immobilized onto Series S Sensor Chips CM5 via amine coupling at 25 °C as described by GE Healthcare, resulting in immobilization values of about 2150 RU.

Techniques: Binding Assay, Modification, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Effect of film modification on secreted protein content in the supernatant and mineralization during osteogenic differentiation of SaOS-2 cells. Cells were seeded on differently modified surfaces at a density of 10,000/cm 2 and differentiated for 10 days. ( A ) Sclerostin, ( B ) Wnt3a, and ( C ) DKK1 were then quantified from the cell supernatant via ELISA after 1, 3, 6, 8, and 10 days. Calcium content ( D ) was quantified at day 10. Film surfaces: ED900: surface coating with ED900 preincubated with soluble sHA3; sHA3: covalent ED900 and sHA3-coating; sHA3 pre: sHA3 surface preincubated with 40 ng/mL sclerostin; w / o cells: sHA3 surface without seeded cells. All values represent the mean ± SD of n ≥ 4. *** p < 0.001 denotes significant difference vs. the respective surface modifications.

Article Snippet: Recombinant human Sclerostin (R&D Systems, Minneapolis, MN, USA) was immobilized onto Series S Sensor Chips CM5 via amine coupling at 25 °C as described by GE Healthcare, resulting in immobilization values of about 2150 RU.

Techniques: Modification, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Effect of film modification on secreted protein content in the supernatant and mineralization during osteogenic differentiation of hMSC. Cells were seeded on differently modified surfaces at a density of 15,000/cm 2 and differentiated for 12 days. ( A ) Sclerostin was then quantified from the cell supernatant via ELISA after 1, 3, 7, 10, and 12 days. OsteoImage TM staining was assessed at day 12 and the fluorescence signal quantitatively ( B ) and qualitatively( C , D ) analyzed. Film surfaces: ED900: surface coating with ED900 preincubated with soluble sHA3; sHA3: covalent ED900 and sHA3-coating; w / o cells: sHA3 surface without seeded cells. All values represent the mean ± SD of n ≥ 4. * p < 0.05; *** p < 0.001 denotes significant difference vs. the respective surface modifications.

Article Snippet: Recombinant human Sclerostin (R&D Systems, Minneapolis, MN, USA) was immobilized onto Series S Sensor Chips CM5 via amine coupling at 25 °C as described by GE Healthcare, resulting in immobilization values of about 2150 RU.

Techniques: Modification, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence

Journal: Nature metabolism

Article Title: Transferrin receptor 2 controls bone mass and pathological bone formation via BMP and Wnt signaling

doi: 10.1038/s42255-018-0005-8

Figure Lengend Snippet: (a-b) Surface plasmon resonance (SPR) experiments using Tfr2-ECD immobilized on the sensor chip and 1 mg/ml holo-transferrin (holo-Tf), different BMP ligands (50 nM), or 200 nM BMP receptors as analytes. All experiments were performed three independent times. (a) Quantification of the binding level normalized to the molecular weight. (BMP-2 n=6, BMP-4, 6, 7, BMPR-II and BMPR-IA n=4 per group, holo-Tf n=3). (b) Binding response of different concentrations of holo-Tf and/or BMP-2. (n=3; ### p<0.001 vs. holo-Tf alone; ***p<0.001 vs. BMP-2 alone). (c) Competitive BMP-2 sandwich ELISA was performed to test Tfr2-ECD (or BMPR-IA as a positive control) binding to BMP-2. A defined concentration of BMP-2 (1,500 pg/ml) was used with increasing concentrations of Tfr2-ECD and BMPR-IA. The principle of the assays is shown in the right schematic. (n=4). (d) SPR analyses. BMP-2 was injected either alone or with increasing concentrations of BMPR-IA. Binding response is relative to BMP-2 alone. (n=4; ### p<0.001 vs. BMPR-IA alone; ***p<0.001 vs. BMP-2 alone). (b, d) One-way ANOVA was used for statistical analysis. (e) HO in 12-week-old female WT and Tfr2 -/- mice after two weeks quantified by µCT (without the bone volume of the tibia). Representative images are shown at the right. (WT PBS n=5, WT Tfr2-ECD n=3, Tfr2 -/- PBS n=4, Tfr2 -/- Tfr2-ECD n=4; *p<0.05; ***p<0.001 to respective PBS control; ### p<0.001 vs. WT). Two-way ANOVA with Bonferroni post-hoc test was used for statistical analysis. (f) HO in 12-week-old female WT mice after two weeks quantified by µCT (without the bone volume of the tibia). (PBS n=12, local Tfr2-ECD n=6, systemic Tfr2-ECD n=8, Pal n=11; *p<0.05 vs. PBS control). (g) Trauma-induced HO model in 12-week-old female WT mice. HO after three weeks assessed using µCT. (n=6 per group; *p<0.05, ***p<0.001 vs. PBS control). (f, g) One-way ANOVA was used for statistical analysis. All data are presented as mean±SD. (h) Scheme of the mode of action of the Tfr2-ECD in the treatment of HO. Left: HO is induced by overactive BMP signaling, leading to the induction of osteoblastic genes and bone formation. Right: Tfr2-ECD neutralizes BMPs, preventing them from activating BMP signaling. Thus, osteoblastic bone formation is inhibited.

Article Snippet: The transferrin saturation was determined using a total iron binding capacity kit from Randox.

Techniques: SPR Assay, Binding Assay, Molecular Weight, Sandwich ELISA, Positive Control, Concentration Assay, Injection

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , Heat map representing hierarchical clustering of differentially expressed Dlk2 gene in dissected zebrafish heads throughout development from 12 hpf to 15 dpf (single-cell sequencing datasets). b , RT–qPCR analysis of mRNA expression of Dlk2 in the developing wild-type embryos. Data are mean of fold changes in relation to mobk13. Data are mean ± s.d ( n ≥ 3 independent biological replicate experiments). **P ≤ 0.01 by unpaired Student’s t -test. c , Representative confocal images of wild-type zebrafish brain at 24 hpf (dorsal view) stained for Dlk2 (green, arrowheads indicating stronger Dlk2 expression) and DAPI (DNA, blue); ( n ≥ 3 independent biological replicate experiments). Scale bars, 100 μm. d , Representative confocal images of wild-type zebrafish telencephalon pallium section at 4 mpf (coronal section) stained for Dlk2 (green), neuronal marker HuC/D (red) and DAPI (DNA, blue); ( n ≥ 3 independent biological replicate experiments). Scale bar, 10 μm. e , Representative confocal images of wild-type (left) and Dlk2 mutant (right) zebrafish brain at 48 hpf (dorsal view) stained for GFAP (red) and DAPI (DNA, blue); ( n ≥ 3 independent biological replicate experiments). Scale bars, 100 μm. f , Western blot of GFAP in zebrafish embryos at 48 hpf, 5 dpf, and 4 mpf in wild-type and Dlk2 mutant. Vinculin and GAPDH were used as loading controls. ( n ≥ 3 independent biological replicate experiments). g, h Representative confocal images of wild-type zebrafish telencephalon: dorso-medial ( g ) and dorso-lateral ( h ) pallium section at 4 mpf (coronal section) stained for GFAP (red), neuronal marker HuC/D (green) and DAPI (DNA, blue). Scale bars, 100 μm. ( n ≥ 3 independent biological replicate experiments).

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Staining, Marker, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , RT–qPCR analysis of wild-type mRNA expression of Dlk2 in the adult (4 mpf) telencephalon. Data are mean of fold changes in relation to actb2. Data are mean ± s.d ( n ≥ 3 independent biological replicate experiments). b , Diagram of Dlk2 zebrafish DNA sequence. Grey boxes represent exons; essential splice site sequences between exon 3 and 4 are shown, with the mutation A>T present in Dlk2 mutant. c , Sanger sequencing chromatograms of the essential splice site intronic sequences before exon 4 of Dlk2 comparing the wild-type with the Dlk2 mutant. d , Genotyping PCR comparing the wild-type with the Dlk2 mutant. e , RT–qPCR analysis of mRNA expression of Dlk2 in the embryo (19 hpf) in wild-type and Dlk2 mutant. Data are mean of fold changes in relation to mobfk13. Data are mean ± s.d ( n ≥ 3 independent biological replicate experiments). ****P ≤ 0.0001 by unpaired Student’s t -test. f , Representative confocal images of 24 hpf wild-type and Dlk2 mutant embryos (wholemount), stained for Dlk2 (green) and DAPI (DNA, blue), ( n ≥ 3 independent biological replicate experiments). g , Representative confocal images of 48 hpf wild-type and Dlk2 mutant embryos (coronal cross section), stained for HuC/D (green), GFAP (red) and DAPI (DNA, blue), ( n ≥ 3 independent biological replicate experiments).

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Quantitative RT-PCR, Expressing, Sequencing, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a, Fiji analysis of GFAP intensity mean in adult telencephalon in wild-type and Dlk2 mutant zebrafish at 4 mpf ( n ≥ 4 independent biological replicate experiments). *P = 0.0425 by unpaired Student’s t -test. b-g Plot representative of the average distance (μm) to the 3 ( a,e ), 5 ( c,f ), 9 ( d,g ) nearest neighbor neuronal cells (HuC/D used as a marker) in wild-type and Dlk2 mutant zebrafish at 4 mpf telencephalon (subpallium). Distribution is compared between the left and right hemispheres as an assessment of symmetry, ( n ≥ 4 independent biological replicate experiments). *P = 0.0130 for 3 nearest neighbor cells, ***P = 0.0009 for 5 nearest neighbor cells, ***P = 0.0005 for 9 nearest neighbor cells, by unpaired Student’s t -test.

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Mutagenesis, Marker

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , Representative confocal images of wild-type (left) and Dlk2 mutant (right) zebrafish brain at 48 hpf (dorsal view) labeled by in situ HCR for Notch1b (red) and DAPI (DNA, blue); ( n ≥ 3 independent biological replicate experiments). Scale bars, 100 μm. b, Representative confocal images of wild-type (left) and Dlk2 mutant (right) zebrafish brain at 48 hpf (dorsal view) labeled by in situ HCR for Her4 (green) and DAPI (DNA, blue); ( n ≥ 3 independent biological replicate experiments). Scale bars, 100 μm . c , Tp1:Venus transgenic Notch reporter expression analyzed by flow cytometry in wild-type (left) and Dlk2 mutant (right) 26 hpf embryos); ( n ≥ 3 independent biological replicate experiments); *P ≤ 0.05 by unpaired Student’s t -test. d, Representative startle experiment in wild-type (left) and Dlk2 mutant (right) zebrafish larvae at 7 dpf; ( n ≥ 3 independent biological replicate experiments, each with n=12 zebrafish per genotype). e , Violin plot representative of the total distance traveled upon stimulus (excluding rest phases) measured in mm during the startle experiment ( d ) ( n ≥ 3 independent biological replicate experiments, each with n=12 zebrafish per genotype). ****P ≤ 0.0001 by unpaired Student’s t -test.

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Mutagenesis, Labeling, In Situ, Transgenic Assay, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a, Representative startle experiment upon light changes through time (grey boxes: light off) in wild-type (left) and Dlk2 mutant (right) zebrafish larvae at 7 dpf; ( n ≥ 3 independent biological replicate experiments, each with n=12 zebrafish per genotype). b, Violin plot representative of the total distance traveled measured in mm during the experiment ( a ) ( n ≥ 3 independent biological replicate experiments, each with n=12 zebrafish per genotype). ****P ≤ 0.0001 by unpaired Student’s t -test

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , Expression level analysis of Dlk2 and Numb using TCGA and GTEX datasets (Gepia2 database) in normal brain tissue (n=30) compared to astrocytoma grade III (n=19), oligodendroglioma grade II (n=38), oligodendroglioma grade III (n=12), and glioblastoma grade IV (n=81). ****P ≤ 0.0001 by unpaired Student’s t -test . b , Fold change expression of notch signaling (receptors and ligands) in glioblastoma grade IV (n=81) TCGA datasets displayed on ( a ). c, Survival analysis by Kapan-Meier curves and log-rank (Mantel-Cox) test in low-grade glioma on the basis of the TCGA database http://www.oncolnc.org , comparing patients’ survival in subgroups high and low for Dlk2 expression, **P = 0.001. d, Co-immunoprecipitation (co-IP) experiment MDA-MB-231 cell lysates that do not express Dlk2. This experiment is in comparison to the co-IP performed in MCF-7 lines that express Dlk2 , to be used as a negative control. Equal amounts of extracts were immunoprecipitated either with Dlk2 antibody or IgG (control). Western blot for input along co-IP samples blotted with Numb antibody. No specific Numb band was detected in the Dlk2 IP compared to the control. ( n ≥ 2 independent biological replicate experiments).

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison, Negative Control, Control, Western Blot

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , Expression level analysis of Dlk2 and Numb using UALCAN web-portal in normal breast tissue (n=114 patients) compared to invasive breast carcinoma (n=1097 patients) (P <1E-12 for Dlk2 as well as Numb). ****P ≤ 0.0001 by unpaired Student’s t -test. b , Kapan-Meier survival curves in invasive breast carcinoma on the basis of the UALCAN database , comparing patients’ survival in subgroups high and low for Dlk2 expression. Survival analysis by Kapan-Meier curves and log-rank (Mantel-Cox) test P=0.0017. c , Western blot of Dlk2 and Numb in MCF-7 and MDA-MB-231 cell lines. GAPDH was used as a loading control. ( n ≥ 3 independent biological replicate experiments). d , Co-immunoprecipitation (co-IP) experiment of Dlk2 in MCF-7 cell lysates. Equal amounts of extracts were immunoprecipitated either with Dlk2 antibody or IgG (control). Western blot for input along co-IP samples blotted with Numb antibody. ( n ≥ 2 independent biological replicate experiments). e , Overexpression experiment of Dlk2 by transfection in MCF-7 cell line. Western blot of Dlk2, Dlk2-Mb (cleaved protein, corresponds to the molecular weight of the membrane-bound fragment) and Vinculin (loading control). Samples labeled as: transfection conditions control (Ctl. Tr), empty vector (E.V. Tr), and Dlk2 transfected cells (Dlk2. Tr). ( n ≥ 3 independent biological replicate experiments). f , Co-immunoprecipitation (co-IP) experiment of Dlk2 in MCF-7 cell lysates comparing control (empty vector, E.V) transfected cells to Dlk2 transfected cells (Dlk2. Tr). Equal amounts of extracts were immunoprecipitated with DKK antibody against the DKK tag present in both the empty vector (control) and Dlk2 transfected cells (Dlk2. Tr). Western blot for input along co-IP samples blotted with Numb antibody. ( n ≥ 2 independent biological replicate experiments). g, h, i., Representative real-time interaction profiles obtained by surface plasmon resonance (SPR) kinetic analyses of the interaction between human Dlk2 extracellular domain (Dlk2EC) and Maltose-binding protein (MBP), negative control ( g ), human Notch1 intracellular domain (NICD) and Numb, positive control ( h ), and Dlk2 and Numb ( i ). The interaction profiles were obtained by binding increasing concentrations of MPB and Numb (0.019 to 20 nM) to DlkEC and NICD immobilized on sensor chips. The kinetic analyses were performed at 25 °C. Each graph represents experimental curves (black lines) and curves obtained by fitting the data using BIAevaluation software (red line). ( n ≥ 2 independent biological replicate experiments).

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Transfection, Molecular Weight, Membrane, Labeling, Plasmid Preparation, SPR Assay, Binding Assay, Negative Control, Positive Control, Software

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a,b,c, Representative real-time interaction profiles obtained by surface plasmon resonance (SPR) kinetic analyses of the interaction between human Dlk2 extracellular domain (Dlk2EC) and different negative controls: Human B7H2 ( a ), Human SNAPC1 ( b ), Human MTERF ( c ). The interaction profiles were obtained by binding increasing concentrations of B7H2, SNAPC1 and MTERF (0.019 to 20 nM) to DlkEC immobilized on sensor chips. The kinetic analyses were performed at 25 °C. Each graph represents experimental curves (black lines); as no interaction was detected, no fitting of the data is presented. d,e, Representative real-time interaction profiles obtained by surface plasmon resonance (SPR) kinetic analyses of the interaction between human Dlk2 extracellular domain (Dlk2EC, residues 1-306) and the negative control Human MBP ( d ) and Human Mdm2 (residues 119-438) ( e ). The interaction profiles were obtained by binding increasing concentrations of MBP and Mdm2 (0.19 to 200 nM) to DlkEC immobilized on sensor chips. The kinetic analyses were performed at 25 °C. Each graph represents experimental curves (black lines) and curves obtained by fitting the data using BIAevaluation software (red line). ( n ≥ 2 independent biological replicate experiments). f,g,h, Representative real-time interaction profiles obtained by surface plasmon resonance (SPR) kinetic analyses of the interaction between human Dlk2 extracellular domain (Dlk2EC) and Human Mdm2 N-ter (residues 1-134) ( f ), Human Mdm2 central domain (residues 134-334) ( g ), and Human Mdm2 C-ter (residues 334-491) ( h ). The interaction profiles were obtained by binding increasing concentrations of Human Mdm2 N-ter, Human Mdm2 central domain, Human Mdm2 C-ter (0.19 to 200 nM) to DlkEC immobilized on sensor chips. The kinetic analyses were performed at 25 °C. Each graph represents experimental curves (black lines) and curves obtained by fitting the data using BIAevaluation software (red line). ( n ≥ 2 independent biological replicate experiments). i, in vitro ubiquitination assay, p53 was incubated with ubiquitin, E1, E2 (UbcH5b), and the presence or absence of Mg-ATP and Mdm2 as indicated. Ubiquitination was detected by Western blot using anti-Mdm2 antibody and anti-p53 antibody ( n ≥ 2 independent biological replicate experiments).

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: SPR Assay, Binding Assay, Negative Control, Software, In Vitro, Ubiquitin Proteomics, Incubation, Western Blot

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , Overexpression experiment of Dlk2 by transfection in MDA-MB-231cell line. Western blot of Dlk2, and Vinculin (loading control). For the samples: transfection conditions control, empty vector (Empty.v), and Dlk2 transfected cells (Dlk2. Tr). ( n ≥ 2 independent biological replicate experiments). b , Cell viability assay of MDA-MB-231 cells comparing empty vector-transfected control cells (Empty.v) with Dlk2-transfected cells (Dlk2. Tr). Using the CellTiter-Glo assay, luminescence (RLU, relative light unit) was measured as an indicator of cell proliferation. Data are mean ± s.d taken every 24 h in replicate wells ( n ≥ 3 independent biological replicate experiments). c , Representative confocal images of MDA-MB-231 cells comparing empty vector-transfected control cells (Empty.v) with Dlk2-transfected cells (Dlk2. Tr) stained for Numb (green) and DAPI (DNA, blue), left; Boxes outline enlarged area displayed on the right panels. Scale bars, 10 μm ( n ≥ 3 independent biological replicate experiments). d, Representative confocal images of MDA-MB-231 cells comparing empty vector-transfected control cells (Empty.v) with Dlk2-transfected cells (Dlk2. Tr) stained for Numb (green), Mdm2 (red) and DAPI (DNA, blue). Scale bars, 10 μm ( n ≥ 3 independent biological replicate experiments). e,f,g, Quantification of immunostainings representative of the confocal images displayed in ( d ). ( n ≥ 114 cells for the empty vector control cells and n ≥ 130 for Dlk2-transfected cells). ****P ≤ 0.0001 by unpaired Student’s t -test. h, Western blot of phosphoSer276 Numb in MCF-7 (left) and MDA-MB-231 (right) cell lines. Vinculin (loading control). ( n ≥ 3 independent biological replicate experiments). i, Western blot of phosphoSer276 Numb in adult zebrafish telencephalon comparing wild-type and Dlk2 mutant. Vinculin (loading control). ( n ≥ 3 independent biological replicate experiments). j,k Representative confocal images of 48 hpf wild-type ( j ) and Dlk2 mutant (Dlk2.mut) ( k ) embryos (coronal cross section), stained for phosphoSer276 Numb (red) and DAPI (DNA, blue). Boxes outline the ventricle at the top of the thalamus in the midbrain. Scale bars, 100 μm ( n ≥ 3 independent biological replicate experiments).

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Over Expression, Transfection, Western Blot, Control, Plasmid Preparation, Viability Assay, Glo Assay, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

doi: 10.1101/2023.07.20.549453

Figure Lengend Snippet: a , Cell viability assay in MCF-7 cells comparing empty vector-transfected control cells (Empty.v) with Dlk2-transfected cells (Dlk2. Tr). Using the CellTiter-Glo assay, luminescence (RLU, relative light unit) was measured as an indicator of cell proliferation. Data are mean ± s.d taken every 24 h in replicate wells ( n ≥ 3 independent biological replicate experiments). b, Overexpression experiment of Dlk2 by transfection in MDA-MB-231 cell line. Western blot of Numb, Dlk2, and Vinculin (loading control). Samples labeled as empty vector (Empty.v) and Dlk2 transfected cells (Dlk2. Tr). ( n ≥ 3 independent biological replicate experiments). c, Protein sequence alignment of Human Numb (P49757) with zebrafish Numb (Q1XD15) using Clustal Omega multiple sequence alignment program. The asterisk symbol indicates positions that have a single, fully conserved residue between the two aligned sequences. The square highlights the alignment with the human Ser276 residue.

Article Snippet: Lipofectamine 3000 reagents (Invitrogen) were used to transfect pCMV6-Entry Mammalian Expression control (PS100001, Origene) and Human Dlk2 pCMV6 (RC210622, Origene) vectors.

Techniques: Viability Assay, Plasmid Preparation, Transfection, Control, Glo Assay, Over Expression, Western Blot, Labeling, Sequencing, Residue

Journal: Frontiers in Immunology

Article Title: Role of N- Glycosylation in FcγRIIIa interaction with IgG

doi: 10.3389/fimmu.2022.987151

Figure Lengend Snippet: Graphic depiction of glycoengineered IgG1 and FcγRIIIa production, validation, and binding studies. (A) Top: Production of CHO WT stably expressing IgG1. In this parental clone, KO and KI of glycosyltransferases resulted in a library of genetically glycoengineered CHO clones stably producing IgG1. Expressed IgG1 was purified by protein G and validated by SDS-PAGE and Mass Spectrometry (MS). Bottom, HEK293 cells were genetically engineered to display distinct N- glycan structures and this library of genetically engineered HEK clones were transiently transfected with soluble HIS- and AviTag-tagged FcγRIIIa-158F/V. Produced FcγRIIIa was purified by Ni-NTA columns and subjected to SDS-PAGE and MS for validation. (B) Schematic depiction of the IBIS MX96 SPR setup. 1) All glycoengineered FcγRIIIa glycoforms were enzymatically biotinylated by BirA and spotted at four different concentrations on a streptavidin-coated chip. 2) Glycoengineered IgG1 was injected at eight different dilutions, 3) allowing for binding affinity measurements of each antibody to all glycoengineered FcγRIIIa’s simultaneously. 4) Regeneration after every sample was carried out after which the next IgG1 glycoform was injected. (C) Glycoengineered IgG1 and FcγRIIIa-158F/V expressing cell lines with clone name, gene editing background and expected N- glycan signature based on gene editing signature and literature. IgG1 has one N- glycan site (Asn297) per Fc domain and FcγRIIIa has 5 N- glycan sites (Asn38, Asn45, Asn74, Asn162, and Asn169). Designations for monosaccharides are according to the Consortium for Functional Glycomics (CFG) .

Article Snippet: The biotinylated, recombinant, human FcγRIIIa-158F and 158V from SinoBiological (10389-H27H-B and 10389-H27H1-B, respectively) were used as an control for SPR.

Techniques: Binding Assay, Stable Transfection, Expressing, Clone Assay, Purification, SDS Page, Mass Spectrometry, Transfection, Produced, Injection, Functional Assay

Journal: Frontiers in Immunology

Article Title: Role of N- Glycosylation in FcγRIIIa interaction with IgG

doi: 10.3389/fimmu.2022.987151

Figure Lengend Snippet: SDS-PAGE of purified recombinant IgG1 and FcγRIIIa-158F/V. Reducing SDS-PAGE of 1 μg purified (A) glycoengineered IgG1 produced in CHO cells with the heavy chain and light chain at ~50 and 30 kDa, respectively. (B) HEK293-expressed His- and AviTag-tagged FcγRIIIa-158F (left lane) and -158V (right lane) migrating as a broad band with a molecular weight ranging from 40 to 50 kDa. Designations for monosaccharides according to the CFG are indicated .

Article Snippet: The biotinylated, recombinant, human FcγRIIIa-158F and 158V from SinoBiological (10389-H27H-B and 10389-H27H1-B, respectively) were used as an control for SPR.

Techniques: SDS Page, Purification, Recombinant, Produced, Molecular Weight

Journal: Frontiers in Immunology

Article Title: Role of N- Glycosylation in FcγRIIIa interaction with IgG

doi: 10.3389/fimmu.2022.987151

Figure Lengend Snippet: Site-specific N- glycan profiling of Asn162 FcγRIIIa-158F/V by LC-MS/MS. FcγRIIIa-158F and -158V expressed in (A) CHO and (B–H) HEK293 cells were digested by a combination of chymotrypsin and Glu-C and subjected to LC-MS/MS. Site-specific N- glycan profiling of Asn162 was carried out and the top 90% of the structures are represented relative to the most abundant structure (A.U. = 1) for FcγRIIIa-158F (dark grey, left) and -158V (light grey, right) with H: hexose, N: N-acetylhexosamine, F: fucose; S: Sialic acid. Proposed glycan structures are based on gene editing signatures and literature. Structures are depicted following the CFG notation .

Article Snippet: The biotinylated, recombinant, human FcγRIIIa-158F and 158V from SinoBiological (10389-H27H-B and 10389-H27H1-B, respectively) were used as an control for SPR.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: Role of N- Glycosylation in FcγRIIIa interaction with IgG

doi: 10.3389/fimmu.2022.987151

Figure Lengend Snippet: IgG1 and FcγRIIIa-158V binding. Surface plasmon resonance dissociation constant (K D ) was determined from SPR analysis after biotinylated glycoengineered FcγRIIIa-158V was spotted at 4 concentrations and bound to glycoengineered IgG1 at 8 times dilution series. Calculation of the dissociation constant was performed by equilibrium fitting to R max = 500. Mean data is reported of three independent experiments for each IgG1:FcγRIIIa-158V pair.

Article Snippet: The biotinylated, recombinant, human FcγRIIIa-158F and 158V from SinoBiological (10389-H27H-B and 10389-H27H1-B, respectively) were used as an control for SPR.

Techniques: Binding Assay, SPR Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: SDS‐PAGE analysis of anti‐HER2 VHH batches. Migration of ∼2 μg purified anti‐HER2 VHH in each lane. MW, molecular weight ladder; Pp, Pichia pastoris expression system; Ec, Escherichia coli expression system; Synth, Chemical synthesis

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: SDS Page, Migration, Purification, Molecular Weight, Expressing

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Schematic representation of the strategy for the chemical synthesis of anti‐HER2 VHH. (a) Anti‐HER2 VHH amino acid sequence. (b) Amino acid sequences of the various peptide segments; X indicates position of the linker RMBO incorporation at the Gly9 residue. (c) Synthetic strategy

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Sequencing

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Analytical data for the characterization of the various peptides of the anti‐HER2 VHH chemical synthesis. (a) HPLC traces and deconvoluted MS for initial peptide segments F1 (blue), F2 (red), and F3 (green). (b) HPLC trace and deconvoluted MS of first purified ligation product F1‐2. (c) HPLC trace and deconvoluted MS of purified ligation product F1‐2‐3. (d) HPLC trace and deconvoluted MS of final product full‐length VHH anti‐HER2

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Purification, Ligation

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Heterogeneity/homogeneity assessment of anti‐HER2 VHH batches by LC‐UV‐MS and native MS. HPLC‐MS analysis in denaturing conditions of anti‐HER2 VHH batches: chemical synthesis batch (a), Pichia pastoris batch (b), and Escherichia coli batch (c) UV chromatograms

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Identification of anti‐HER2 VHH species by liquid chromatography coupled to mass spectrometry

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Liquid Chromatography

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Native mass spectrometry analysis of anti‐HER2 VHH batches. ESI mass spectra on the m/z range (1,000–3,500) of chemical synthesis (a), Pichia pastoris (b), and Escherichia coli (c) batches in native conditions obtained on a Q‐TOF instrument. (a) (A) 14,022.4 ± 0.4 Da: VHH WT (7+/2,004.1 m/z); (*)14,085.1 ± 0.4 Da (+63 Da): VHH WT (+noncovalent adduct). (b) (B) 14,166.4 ± 0.5 Da: VHH WT (7+/2,024.9); (*) 14,229.2 ± 0.4 Da (+63 Da): VHH WT (+noncovalent adduct). (c) (C) 14,155.1 ± 0.4 Da: VHH WT (7+/2,023.1 m/z); (#) 14,183.2 ± 0.5 Da (+28 Da): VHH WT (+fMet); (d) 13,607.7 ± 0.5 Da: C‐ter truncated VHH (1–123) (7+/1,944.7 m/z); (#)13,635.7 ± 0.5 Da (+28 Da): C‐ter truncated VHH (+fMet). (*) and (#) represent a noncovalent adduct and a formylation, respectively

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Mass Spectrometry

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Native IM‐MS experiments for conformational characterization of anti‐HER2 VHH batches. ATDs of the 7+ charge state of the three VHHs at low collision energy (4 V)

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Gas‐phase conformational stability of anti‐HER2 VHH batches by collision induced unfolding experiments. CIU plots of anti‐HER2 VHH batches. CIU fingerprints of the 7+ charge state for the VHH from chemical synthesis (a), Pichia pastoris (b), and Escherichia coli (c) batches from 0 to 120 V collision energies. ATD extraction of the 7+ charge state of the three VHHs at 35 V collision energy (d)

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Gas‐phase conformational stability of nonreduced and reduced anti‐HER2 VHH produced by Pichia pastoris. CIU plots of the 7+ charge state for the nonreduced (a) and reduced (b) anti‐HER2 VHH samples

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Produced

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Functional characterization of VHH‐antigen interaction by SPR. Panels A to C: Calibration‐free concentration analysis of VHH active concentration. CFCA fits for the binding between highly immobilized HER2 antigen (1,200 RU) and a VHH dilution (~1 nM) tested in duplicates at two different flow rates (10 and 80 μL.min−1). The VHH samples were prepared from (a) Escherichia coli periplasmic expression, (b) chemical synthesis, and (c) Pichia pastoris secretion. The figures show overlay plots of experimental (black) and calculated (red) data. Calculated concentration in nM, closeness of the fit (chi2) and degree of mass transport limitation (QC ratio) for each sample: (a) 0.85 nM, chi2 = 0.022, QC ratio = 0.691; (b) 0.99 nM, chi2 = 0.047, QC ratio = 0.662; (c) 0.96 nM, chi2 = 0.011, QC ratio = 0,718. Panels (d)‐(g) Sensorgrams of the interaction between immobilized HER2 antigen (100 RU) and increasing concentrations of VHH (0.33, 1, 3.33, 10, 30 nM, duplicates). (d) Escherichia coli periplasmic expression, (e) chemical synthesis, (f) Pichia pastoris secretion, and (g) anti‐ADORA2A VHH, negative control. For (d), (e), and (g), experimental curves (black) were fitted with a 1:1 Langmuir binding model (red) using the scrubber 2.0c software, residual plots obtained in global analysis of each binding curves are shown and are randomly distributed

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Functional Assay, Concentration Assay, Binding Assay, Expressing, Negative Control, Software

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Binding parameters of anti‐HER2 VHH to its antigen HER2 determined by SPR ( n = 2)

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Binding Assay, SPR Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

doi: 10.1002/pro.3712

Figure Lengend Snippet: Percentage of active anti‐HER2 VHH in each batch determined by SPR

Article Snippet: The his‐tagged N‐terminal extracellular domain of human HER2 protein (Sino Biological, Cat.

Techniques: Protein Concentration

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: Production and characterization of the ALCAM-blocking antibody I/F8-Fc. (A) Schematic representation of the homodimeric I/F8-Fc antibody, consisting of scFv IF/8 fused to the Fc portion of murine IgG 1 . (B) SDS-PAGE of I/F8-Fc under non-reducing (nr) and reducing (r) conditions. Expected molecular weight: 51 kDa under reducing conditions. (C) In FPLC I/F8-Fc elutes as a homodimer. (D,E) Surface plasmon resonance profiles of purified I/F8-Fc on micro sensor chips coated with (D) human and (E) murine ALCAM. (F,G) FACS analysis reveals I/F8-Fc binding to (F) human and (G) murine endothelial cell lines. (H–J) Analysis of I/F8-Fc specificity by FACS analysis performed on single cell suspensions generated from murine lungs of WT and ALCAM −/− mice. (H) FACS gating strategy for differentiating BECs from LECs. Single-cell suspensions from enzymatically digested lung tissue were stained for CD45, CD31, and podoplanin. BECs and LECs were identified, by gating on CD45 − CD31 + podoplanin − (BECs) or CD45 − CD31 + podoplanin + (LECs) cells. (I,J) I/F8-Fc stains BECs (CD45 − CD31 + podoplanin − ) and LECs (CD45 − CD31 + podoplanin + ) in lung single-cell suspension derived from (I) WT mice but not from (J) ALCAM −/− mice. Black lined histogram: I/F8-Fc, shaded histogram: KSF-Fc control. Representative data from one out of four similar experiments are shown in (F–J) .

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Blocking Assay, SDS Page, Molecular Weight, SPR Assay, Purification, Binding Assay, Generated, Staining, Suspension, Derivative Assay, Control

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: I/F8-Fc reduces (lymph)angiogenic processes and T cell activation in vitro . (A–E) Effects on in vitro (lymph)angiogenis. (A,B) A cell-free scratch was introduced into confluent monolayers of (A) human LECs or (B) HUVECs and the impact of I/F8-Fc or KSF-Fc control antibody on VEGF-A-induced scratch closure was analyzed after 24 and 12 h, respectively (C) Blocking ALCAM with I/F8-Fc reduced tube formation of human LECs. (D,E) A cell-free scratch was introduced into confluent monolayers of (D) murine MS-1 cell or (E) murine primary dermal LECs and the impact of I/F8-Fc on scratch closure was analyzed after 24 and 27 h, respectively. Data from 1 out of 3 to 4 similar experiments are shown in (A–E) . (F–I) Effects on in vitro T cell activation. WT or ALCAM −/− BM-DCs were pulsed with OVA peptide in presence of LPS and co-incubated with CD4 + OTII cells in presence of I/F8-Fc or KSF-Fc control antibody. (F) FACS analysis demonstrating ALCAM and CD6 expression in BM-DCs and OTII cells, respectively. (G,H) Impact of I/F8-Fc treatment on T cell proliferation. (G) Representative FACS plots showing CFSE-dilution, as a readout of T cell proliferation. (H) Quantitation of proliferating cells. (I) T cell-mediated IFN-γ production was quantified in the cell culture supernatants. Data from 1 out of 4 similar experiments ( n = 6 replicates) are shown in F-I. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Activation Assay, In Vitro, Control, Blocking Assay, Incubation, Expressing, Quantitation Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: I/F8-Fc treatment reduces developmental lymphangiogenesis in the diaphragm to a similar degree as ALCAM deficiency. (A) Schematic representation of the diaphragm and of the area analyzed by LYVE-1 immunofluorescence in D-J. TW: thoracic wall; CT: central tendon. (B) ALCAM expression on diaphragmatic P5 BECs (CD45 − CD31 + podoplanin − ) and LECs (CD45 − CD31 + podoplanin + ) was confirmed by FACS. Data from 1 out of 2 similar experiments are shown. Specific stainings are shown as black lined and isotype control stainings as gray tinted histograms. (C) Schematic presentation of the antibody treatment protocol. Pregnant mice received I/F8-Fc or KSF-Fc antibody i.p. (300 μg) on day E15.5 and E17.5 (indicated by an asterisk). In addition, newborn mice received antibody i.p. (30 μg) on day P1 and P3 (indicated by a circle). (D–H) On P5 the diaphragm was collected and lymphatic vessels were visualized by LYVE-1 staining. (D,E) Representative stainings from diaphragms of KSF-Fc- or I/F8-Fc-treated animals. (F–H) Image-based morphometric analysis of (F) the LYVE-1 + area, (G) the number of branch points and (H) the total vessel length in the diaphragm of I/F8-Fc or KSF-Fc control-treated pups. Each dot represents the measurement made in one animal ( n = 10). Data from 1 out of 3 similar experiments are shown in F-H. (I,J) Image-based morphometric analysis of (I) the number of branch points and (J) the total vessel length in the P5 diaphragm of WT and ALCAM −/− mice. Data from 1 experiment ( n = 6 or 9 mice) are shown in G-H. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Immunofluorescence, Expressing, Control, Staining

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade reduces DC transmigration and crawling on lymphatic endothelium in vitro . (A,B) imLECs were seeded on the lower surface of transwell inserts and grown to confluence. Transmigration of BM-derived WT or ALCAM –/– BM-DCs across imLEC monolayers was investigated in presence of either KSF-Fc (control) or I/F8-Fc antibody. (A) Absolute numbers and (B) % of transmigrated DCs from the input. Each dot represents the value obtained from one transwell. Pooled data from 3 similar experiments (involving 3 transwells per condition per experiment) are shown in (A,B) . (C) DCs isolated from either WT or ALCAM −/− mice were treated with I/F8-Fc or KSF-Fc antibody, followed by analysis of crawling speed on ALCAM-expressing imLEC monolayers. Each dot represents one DC. Pooled data from 2 to 3 similar experiments are shown. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Transmigration Assay, In Vitro, Derivative Assay, Control, Isolation, Expressing

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade reduces corneal allograft rejection. (A) Schematic description of the corneal allograft rejection model. On day −14 three sutures were placed into the corneas of recipient BALB/c mice. On day 0, sutures were removed and the donor corneas from C57BL/6 mice were transplanted. Mice received I/F8-Fc or KSF-Fc control antibody i.p. (150 μg) on day −1, 2, 5, 8, and 12 (indicated by an asterisk). Additionally, mice were treated by topical application of antibody (3 μg) twice a day for 14 days (day 3–14; gray bar). Transplant survival was monitored over 8 weeks (day 0–56). Transplants were considered rejected when the cornea lost its transparency. (B) Representative images of a surviving and a rejected cornea. The graft border is indicated by an asterisk. (C) Impact of antibody treatment on corneal allograft survival. NR group: normal risk group, consisting of mice that received a cornea transplant without the previous induction of suture-induced inflammation. Statistical analysis: I/F8-Fc vs. KSF-Fc: p = 0.024; I/F8-Fc vs. NR: p = 0.77; KSF-Fc vs. NR: p = 0.019. (D–F) FACS analysis was performed on eye-draining cervical LNs of all mice at the end of the study. Percentage of (D) CD45 + CD3 + T cells, (E) CD45 + CD11c + DCs (F) and CD45 + CD4 + Foxp3 + Tregs in cervical LNs. Pooled data from 2 experiments with a total of 10 mice /group are shown.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade results in accumulation of corneal DCs. (A) Schematic description of the suture model. Three sutures were placed into the corneas. Mice received I/F8-Fc or KSF-Fc control antibody i.p. (150 μg) on day 2, 5, 8, and 12 (indicated by an asterisk) as well as antibody-containing eye drops (3 μg) twice a day for 14 days (gray bar). Corneas were analyzed by whole-mount immunofluorescence on day 14 ( n = 5/group). (B–G) Representative immunofluorescence images and quantifications of the percentage of corneal area covered with (B,C) LYVE-1 + lymphatic vessels (LVs), (D,E) CD31 + blood vessels (BVs), scale bars: 500 μm, and (F,G) CD11c + DCs, scale bars: 100 μm. Images on the left in (F) show CD11c + cells (green) detected by immunofluorescence. Images on right show corresponding cells (red) identified by gray-image threshold analysis, which was used for quantification (G) . (H) Schematic representation of the suture/allograft model: three sutures were placed into the corneas and animals were treated as in (A) . On day 14 corneal transplantation was performed. Five days after transplantation (day 19), corneas were analyzed by whole-mount immunofluorescence ( n = 10/group). (I–L) Representative images and quantifications of the percentage of corneal area covered with (I,J) CD11c + DCs and (K,L) CD3 + T cells. DC and T cell quantification in (I–L) was performed as in (F,G) . Images on the left in (I,K) depict DC and T cells as seen in immunofluorescence; images on the right (in red) as identified by gray-image threshold analysis. Scale bars: 100 μm.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Control, Eye Drops, Immunofluorescence, Transplantation Assay

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade reduces DC emigration from human skin. Human skin sections were stained for (A) LYVE-1 and (B) MHCII, in combination with either an ALCAM-specific or the corresponding isotype control antibody. ALCAM reactivity was observed in (A) all LYVE-1 + vessels and (B) in some MHCII + cells. Scale bars: 50 μm. (C) Human skin punch biopsies were incubated in culture medium and the presence of ALCAM on emigrated CD86 + MHCII hi cells was analyzed after 24 h. Data from 1 representative of a total of 3 experiments are shown. (D) Human skin DC emigration experiment. Punch biopsies of human skin were incubated in culture medium in presence of either I/F8-Fc or of KSF-Fc control antibody, and cells emigrated into the culture medium were analyzed by FACS 24 h later. In (C) representative FACS plots show the gating scheme. Migratory DCs were identified as CD86 + MHCII hi cells and were quantified with the help of counting beads. Each dot represents one biopsy ( n = 10 per condition).

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Staining, Control, Incubation

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM is expressed in pathologic blood and lymphatic vessels in neovascularized human corneas and in corneal DCs. Serial sections of inflamed human corneas obtained from high risk transplant patients were stained for (A) ALCAM and LYVE-1 and (B) ALCAM and MHC-II. Magnifications of the boxed areas are shown in the center part of the Figure. LV, Lymphatic vessel; BV, Blood vessel, E, epithelium. Arrows indicate specific staining. Scale bar: 20 μm. Representative images from 3 stained neovascularized corneas are shown.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Staining