Journal: Journal of Immunological Methods

Article Title: Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects

doi: 10.1016/j.jim.2017.01.008

Figure Lengend Snippet: Depletion of IL-10 from ascites using αIL-10 neutralizing antibody and Protein G-coated agarose beads. (A) Levels of mouse IgG2b αIL-10 neutralizing antibody in ascites samples were measured after depletion of IL-10 using Protein G-coated agarose beads. IL-10 depletion was performed with 5 μg/ml neutralizing antibody (lower panel) in two ( n = 2) and with 20 μg/ml neutralizing antibody (top panel) in four ( n = 4) ascites samples. The mouse IgG2b antibody concentration was determined by sandwich ELISA. (B) Human IgG levels in ascites were measured by sandwich ELISA before (control) and after depletion with Protein G-coated agarose beads (depleted). The two experiments where 5 μg/ml neutralizing antibody was used are shown as dashed lines ( n = 2), while in the remaining experiments ( n = 4) shown as solid lines, a concentration of 20 μg/ml neutralizing antibody was used. Wilcoxon matched pairs test, two-tailed. * = p < 0.05 (C) Schematic depiction of the pitfalls encountered with this experimental approach. (1) Neutralizing αIL-10 mouse IgG2b antibody is added to ovarian carcinoma associated ascites, which contains a high concentration of human IgG. Upon incubation, the neutralizing αIL-10 antibody binds IL-10 present in ascites. (2) Protein G-coated agarose beads are added to the sample. Human IgG competes with IL-10-specific mouse IgG2b antibody for Protein G-binding sites. (3) Centrifugation creates a pellet of agarose beads with predominantly bound human immunoglobulins, leaving IL-10-specific antibody in form of immune complexes with IL-10 or in form of free antibody not bound to its antigen behind in the ascites.

Article Snippet: After washing twice, standard (mouse IgG1 clone 11,711 or mouse IgG2b clone 20,116; both R&D Systems) and samples were added to triplicate wells at appropriate dilutions in PBS.

Techniques: Concentration Assay, Sandwich ELISA, Control, Two Tailed Test, Incubation, Binding Assay, Centrifugation

Journal: Journal of Immunological Methods

Article Title: Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects

doi: 10.1016/j.jim.2017.01.008

Figure Lengend Snippet: Depletion of IL-10 from ascites using Protein G magnetic beads coated with αIL-10 neutralizing antibody. (A) Levels of mouse IgG2b αIL-10 neutralizing antibody in ascites samples were measured after depletion of IL-10 using Protein G coated magnetic beads. The depletion was performed with 1 h ( n = 1) and 16 h ( n = 3) incubation periods for αIL-10 antibody-coated beads in ascites. The mouse IgG2b concentration was quantified by sandwich ELISA. (B) Human IgG levels in ascites were measured by sandwich ELISA before (control) and after depletion with Protein G-coated magnetic beads (depleted). Values before and after 16 h incubation of ascites with antibody-coated Protein G beads are connected by a solid line, while the dashed line indicates human IgG values in ascites before and after an incubation of 1 h. Wilcoxon matched pairs test, two-tailed; ns = not significant. (C) Schematic illustration of the drawbacks of this experimental method. (1) Protein G-coated magnetic beads are incubated with mouse IgG2b αIL-10 neutralizing antibody. The antibody binds to Protein G beads with high affinity. (2) The beads pre-coated with αIL-10-specific antibody are added to ascites to allow binding of IL-10. Partial displacement of mouse IgG2b αIL-10 neutralizing antibody and/or mouse IgG2b control antibody is observed. (3) After removal of Protein G magnetic beads and bound immunoglobulins with the help of a magnet, αIL-10 antibody either with or without bound IL-10 as well as the isotype control antibody are left behind in the ascites.

Article Snippet: After washing twice, standard (mouse IgG1 clone 11,711 or mouse IgG2b clone 20,116; both R&D Systems) and samples were added to triplicate wells at appropriate dilutions in PBS.

Techniques: Magnetic Beads, Incubation, Concentration Assay, Sandwich ELISA, Control, Two Tailed Test, Binding Assay

Journal: Journal of Immunological Methods

Article Title: Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects

doi: 10.1016/j.jim.2017.01.008

Figure Lengend Snippet: Successful depletion of IL-10 from ovarian carcinoma associated ascites using NHS magnetic beads for investigation of paracrine effects. Schematic display of IL-10 depletion with αIL-10 antibody covalently bound to magnetic beads. (1) Magnetic NHS ester beads are biochemically linked to mouse IgG2b αIL-10 neutralizing antibody or isotype control antibody. The bonds between the NHS ester group on the beads and free amines of the antibody are covalent, permanently immobilizing the antibody on the bead surface. (2) Antibody-coated beads are added to ascites and incubated overnight to allow specific binding of IL-10 to its neutralizing antibody. (3) A magnet is used to remove complexes of magnetic beads, αIL-10 neutralizing antibody and IL-10. (B) Human IgG levels in ascites were measured by sandwich ELISA before (control) and after depletion αIL-10 antibody bound to magnetic NHS beads (IL-10 depleted). n = 4; Wilcoxon matched pairs test, two-tailed; ns = not significant. (C) IL-10 levels in depleted versus non-depleted control or mock-depleted ascites sample were measured by FlowCytomix bead ELISA ( n = 1). The IL-10 level in the depleted sample (6.41 pg/ml) falls below assay sensitivity (27 pg/ml). (D) Monocyte-derived DC were stimulated with 1 μg/ml LPS in the presence or absence of 10% ascites from ovarian carcinoma patients which had previously been depleted of IL-10 (IL10-ΔAF) or which had undergone mock depletion (mock-ΔAF). Alternatively, DC cultures were treated with LPS and untreated ascites fluid (AF) in the presence of αIL-10 neutralizing antibody (5 μg/ml); n = 3 (3 independent experiments; monocyte-derived DC from 1 healthy donor, cultured with three ascites samples from three individual ovarian carcinoma patients). IL-6 levels were measured in cell culture supernatants by sandwich ELISA. One-way ANOVA (Friedman test with Bonferroni post-test); *** p < 0.001. (E) As in D; n = 6 (monocyte-derived DC from 3 healthy donors, cultured with two ascites samples from two individual ovarian carcinoma patients). Wilcoxon matched pairs test, two-tailed; ** p < 0.01.

Article Snippet: After washing twice, standard (mouse IgG1 clone 11,711 or mouse IgG2b clone 20,116; both R&D Systems) and samples were added to triplicate wells at appropriate dilutions in PBS.

Techniques: Magnetic Beads, Control, Incubation, Binding Assay, Sandwich ELISA, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture

Journal: Journal of Immunological Methods

Article Title: Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects

doi: 10.1016/j.jim.2017.01.008

Figure Lengend Snippet: Covalent binding of αIL-10 neutralizing antibody to NHS coated magnetic beads leads to minimal residual αIL-10 antibody in depleted ascites samples.

Article Snippet: After washing twice, standard (mouse IgG1 clone 11,711 or mouse IgG2b clone 20,116; both R&D Systems) and samples were added to triplicate wells at appropriate dilutions in PBS.

Techniques: Binding Assay, Magnetic Beads

Journal: Journal of Immunological Methods

Article Title: Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects

doi: 10.1016/j.jim.2017.01.008

Figure Lengend Snippet: Covalent binding of αPGE 2 neutralizing antibody to NHS magnetic beads leads to minimal residual αPGE 2 antibody in depleted ascites samples.

Article Snippet: After washing twice, standard (mouse IgG1 clone 11,711 or mouse IgG2b clone 20,116; both R&D Systems) and samples were added to triplicate wells at appropriate dilutions in PBS.

Techniques: Binding Assay, Magnetic Beads

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID; 37 proteins identified as RNA binding in terms of molecular function are listed in Dataset . ( B ) Interactions of Derlins with Sec61β. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C ) Endogenous interaction of Derlin-1 with Sec61β. Immunoprecipitation (IP) with anti-Sec61β antibody or control (Ctrl) IgG using Protein G Sepharose and immunoblotting (IB) with indicated antibodies in HepG2 cells treated with or without 200 nM Tg and/or 500 nM MG132 for 16 h. ( D – F ) IB of ERpQC substrates in HEK293 cells transfected with indicated siRNAs and plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Black arrowhead, signal peptide-uncleaved NHK QQQ ( S NHK QQQ ); white arrowhead, signal peptide-cleaved NHK QQQ ( C NHK QQQ ). ( G ) IB of ERpQC substrate in wild-type (WT) or Derlin-1, -2 , and -3 triple knockout (TKO) HEK293 cells transfected with indicated siRNAs and plasmid for NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Expression levels of S NHK QQQ were calculated and shown as the percentage of S NHK QQQ out of the total amount of NHK QQQ ( S NHK QQQ and C NHK QQQ ). Black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Mouse IgG2b isotype control monoclonal antibody , Proteintech , Cat. #66360-3-Ig; RRID: AB_2881740.

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot, Triple Knockout, Plasmid Preparation, Expressing

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Volcano plots of the quantitative proteomic analysis of ER stress-induced Sec61β interactome. Plots indicate the fold change in the abundance of identified proteins in Tg and MG132-treated samples compared with that in DMSO-treated samples (log2 fold change [Tg_MG132/DMSO], x axis) against its significance (−log10 P value, y axis) ( n = 4). Orange, significant fold change (Tg_MG132/DMSO) ≥ 2, P value ≤ 0.05; red, ARIH1. See also Dataset . ( B ) Domain structures of human ARIH1 and truncated forms with or without mutation. UBA-L, ubiquitin-associated domain-like; RING1, RING domain 1; IBR, in-between RING domain; RING2, RING domain 2; C357S (CS), catalytically inactive serine mutant of Cys357, the active site of Ub ligase activity; ΔAri, mutant lacking inhibitory Ariadne domain. ( C ) Endogenous interaction of Sec61β with ARIH1 during ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in WT or 3× Flag-tagged Sec61β knock-in HEK293 cells and treated with or without 50 nM Tg and/or 200 nM MG132 for 16 h. ( D ) Interaction of endogenous Sec61β and Derlin-1 with exogenous ARIH1 during ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with Flag-ARIH1 and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-ARIH1 were normalized by the amount of input for each protein and shown as fold increases relative to the control lane. ( E ) Endogenous interaction of Sec61β and ARIH1 with Derlin-1 during ER stress. Wild-type C57BL/6 J mice (12 to 14-week-old), matched for sex, were given a single 2 μg/gram body weight intraperitoneal injection of a 0.1 mg/ml suspension of tunicamycin (Tun) in PBS or vehicle (PBS) alone. After 20 h, mice were deeply anesthetized and transcardially perfused with PBS. Whole cell lysates were prepared by homogenizing livers for 60 s × five times in lysis buffer. Cell lysates were IPed with anti-Derlin-1 antibody or control IgG using Protein G Sepharose. All samples were immunoblotted with indicated antibodies. ( F , G ) Interaction of endogenous ARIH1 ( F ), Sec61β ( F , G ), or Derlin-1 ( F , G ) with exogenous 4EHP in HEK293 cells ( F ) and ARIH1-deficient HEK293 cells ( G ). IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated siRNAs ( G ) and Flag-4EHP ( F , G ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-4EHP were normalized by the amount of input for each protein and shown as fold changes relative to the control lane. ( H , I ) Interaction of endogenous Derlin-1 with exogenous eIF4E in ARIH1- ( H ) or Sec61β- ( I ) deficient cells. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated siRNAs and Flag-eIF4E and treated with 50 nM Tg and 200 nM MG132 for 16 h. The amount of co-IPed Derlin-1 with Flag-eIF4E was normalized by the amount of input Derlin-1 and shown as fold increase relative to the control lane. .

Article Snippet: Mouse IgG2b isotype control monoclonal antibody , Proteintech , Cat. #66360-3-Ig; RRID: AB_2881740.

Techniques: Mutagenesis, Ubiquitin Proteomics, Activity Assay, Knock-In, Transfection, Control, Injection, Suspension, Lysis

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Proteasome chymotrypsin-like peptidase activity of cell extracts from HepG2 cells transfected with siCtrl or siSec61β (#1, #2, or #3) and treated with 2 μg/ml tunicamycin (Tun) for 16 h was measured using Suc-LLVY-AMC as a substrate. Fluorescence intensity was normalized to cell viability in each condition. Proteasome activity is shown as fold decrease relative to that of siCtrl-transfected cells ( n = 7, 7, 6, and 7 from left to right, respectively). ( B ) Degradation of CL1 degron chased for the indicated periods after 15 min pulse of [ 35 S]-methionine/cysteine metabolic labeling shown in Fig. . Cell lysates of HEK293 cells transfected with indicated siRNAs and Venus-CL1-Flag and stimulated with 50 nM Tg for 16 h were IPed with an anti-Flag antibody, resolved by SDS-PAGE and analyzed by autoradiography. ( C ) Representative fluorescence images of HepG2 cells transfected with siSec61β and stimulated with 50 nM Tg for 6 h, followed by staining with ProteoStat (red, protein aggregation), calnexin (green, ER membranes) and DAPI (blue, nuclei). Scale bars, 25 μm. ( D ) Reduced expression of Sec61β in zebrafish by injection of antisense morpholino oligonucleotide (MO). Antisense MO was designed as described in Methods. MOs were injected into zebrafish embryos at 1- to 2-cell stages. The expression of Sec61β in zebrafish at 3 dpf was analyzed by IB using a polyclonal antibody against a peptide against zebrafish Sec61β (SAGTGGMWRFYTEDSPGLKV) raised in rabbits. Lysates were prepared by homogenizing 3 dpf fish for 60 s in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EGTA, and 1% Triton X-100) supplemented with 5 μg/mL leupeptin (Nacalai Tesque; 43449-62) on ice using a Micro Smash (TOMY; MS-100) (4500 rpm, 4 °C). Lysates were resolved by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBS-T (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.05% Tween-20), the membranes were probed with antibody against to zebrafish Sec61β (1/1000) or actin (1/5000) diluted in 5% BSA in TBS-T overnight at 4 °C. Secondary antibodies [IRDye 800CW Donkey anti-Rabbit IgG (H + L) (1/10,000) and IRDye 680RD Donkey anti-Mouse IgG (H + L) (1/10,000)] were diluted in 5% skim milk in TBS-T, and membranes were incubated 2 h at room temperature. Images were revealed and analyzed using Odyssey CLx (LICOR) and Empiria Studio software 3.0 (LICOR). ( E ) Representative images showing the typical morphology of zebrafish larvae injected with water, Sec61β-atg MO or Sec61β-5mis MO at 3 dpf. Arrowhead indicates the abnormal morphology observed in rare Sec61β-deficient zebrafish. ( F ) Histogram showing the average body length of zebrafish larvae relative to that of water-injected controls at 3 dpf. Sec61β-deficient zebrafish ( n = 13) showed a tendency toward reduced body length compared to control groups ( n = 10 for water injection or n = 24 for Sec61β-atg-5mis MO injection). ( G ) The behavior of zebrafish was observed at 4 dpf, and a phenotype scores were recorded manually. The scoring criteria were defined as follows: no obvious abnormality (0), abnormal swimming with head shaking and slightly smaller size (1), abnormal swimming and morphology (2), and no swimming and abnormal morphology (3). ( H ) Exogenous expression of ARIH1 mutants in zebrafish by injection of synthesized mRNAs. Synthesized mRNAs for human ARIHΔAri(CC) or (CS) were co-injected into zebrafish embryos at 1- to 2-cell stages. Lysates were prepared by homogenizing 3 dpf 10 fish for 60 s in lysis buffer due to weak expression of exogenous proteins. The expression of HA-ARIHΔAri was analyzed by IB using a rat monoclonal antibody against HA (clone 3F10) and secondary antibody (HRP-linked anti-rat IgG antibody). The membranes were detected by an ECL system, and images were revealed and analyzed using ChemiDoc Touch (BioRad). Data are means ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siSec61β ( A ); Kruskal–Wallis rank-sum test ( F ).

Article Snippet: Mouse IgG2b isotype control monoclonal antibody , Proteintech , Cat. #66360-3-Ig; RRID: AB_2881740.

Techniques: Activity Assay, Transfection, Fluorescence, Labeling, SDS Page, Autoradiography, Staining, Expressing, Injection, Lysis, Blocking Assay, Incubation, Software, Control, Synthesized, Two Tailed Test

Journal: mAbs

Article Title: Maximizing the potency of an anti-TLR4 monoclonal antibody by exploiting proximity to Fcγ receptors

doi: 10.4161/19420862.2014.975098

Figure Lengend Snippet: Analyses of the various antibody formats ( A ) To assess the purity and confirm the size of each reagent, the various antibody formats were analyzed by Agilent 2100 bioanalyzer. Proteins were assessed under non-reduced (NR) and reduced conditions (R). 1, IgG; 2, heavy chain; 3, light chain; 4, F(ab)’ 2 ; 5, VH-CH1-hinge; 6, monovalent antibody; 7, hinge-Fc; 8, Fab; 9, light chain and VH-CH1. ( B ) The binding of Hu 15C1 (black circles), monovalent Hu 15C1 (gray triangles), F(ab)’ 2 Hu 15C1 (open circles) and Fab Hu 15C1 (open diamonds) to TLR4 was analyzed by competitive ELISA. To compare the different antibody formats, the same number of binding site was used, i.e., the molar concentration of monovalent and Fab is twice the molar concentration of IgG and F(ab)’ 2 . Results are normalized and expressed as mean ± SD of duplicates. An F test was used to compare the fitted curves of different groups. ns: not significant.

Article Snippet: Human IgG1, κ, mouse IgG1, κ and mouse IgG2b, κ isotype control were from Novimmune.

Techniques: Binding Assay, Competitive ELISA, Concentration Assay