Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: (A) S100A9 in the biotinylated plasma membrane fraction of THP-1 cells: non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b). (B) S100A8 expression in biotinylated THP-1 cells (same sample fractions as in (A). (C) Expression of S100A9 (left) and S100A8 (right) in the non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b) of human monocytes. Antibodies used were; α-S100A9 (NOVUS clone 1C10) and α-S100A8 (BMA; T1030; clone 8-5C2). 4 ng recombinant hS100A9 (rA9) was loaded.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Clinical Proteomics, Membrane, Expressing, Recombinant

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: (A) Binding of anti-S100A8 (8-5C2), S100A9 (43/8) and S100A8/S100A9 (27E10) to SA captured biotinylated plasma membranes. Antibodies were injected at 6.25–50 nM for 2 min at 30 μL/min in assay buffer (HBS-P with 1 mM Ca ++ + 20 μM Zn ++ ). A 15 μL pulse of 10 mM glycine-HCl was used for regeneration. Capture level ~ 2.1 kRU. Kinetic evaluation was made using a 1:1 model for mAb 43/8 at 25 and 50 nM. An affinity of 7.0 nM ( k on 1.9 × 10 5 1/Ms; k off 1.3 × 10 −3 1/s) was calculated with maximal binding of 46 RU. (B) Injection of non-biotinylated THP-1 lysate over immobilized mAb 8-5C2, 43/8 and 27E10 (density ~ 3 kRU). Lysate, 0.45 mg/mL, diluted 5 to 40 times in assay buffer, injected for 3 min at 20 μL/min. Distinct binding was obtained when the THP-1 lysate was injected over the 27E10 surface whereas no binding was observed when lysate was passed over 8-5C2 and 43/8. (C) Binding of ten-fold diluted non-biotinylated THP-1 lysate to mAb 27E10 is inhibited after pre-incubation with 50 nM 27E10 but not with 8-5C2 or 43/8. Binding was expressed as % of response without antibody. (D) Sensorgrams obtained after injection of 50 nM mAb 8-5C2, 43/8 and 27E10 over SA captured (level ~ 1.1 kRU) solubilized membranes from surface biotinylated human monocytes. An affinity of ~ 6.1 nM (( k on 2.7 × 10 5 1/Ms; k off 1.6 × 10 −3 1/s) and maximal binding at ~ 12 RU was obtained after fit of sensorgram obtained with the 43/8 antibody to a 1:1 model. (E) Interaction of non-biotinylated lysate from human monocytes with 8-5C2, 43/8 and 27E10. Serially diluted lysate (5- to 40-fold in assay buffer) was injected over immobilized antibodies as in . Binding at high levels was demonstrated to mAb 27E10 (reaching steady state levels at ~ 1,000 RU) and was completely blocked when lysate (diluted 5-fold) was pre-incubated with 50 nM 27E10 but not with 8-5C2 or 43/8 (Fig 2C). Specific (i.e. displaceable by pre-incubation with anti-S100A9; data not shown) binding at much lower levels was also shown to 43/8 whereas non-specific binding was demonstrated to 8-5C2 (Fig 2E).

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Binding Assay, Clinical Proteomics, Injection, Incubation

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: THP1 cells were cultured for 48h in presence or absence of 10ng/ml TNFα and stained with antibodies against S100A9 (purple) (A). THP-1 cells were treated with either TNFα (B) or TNFα + IL10 (C); stained with anti-S100A8 (red), anti-S100A9 (purple) and anti-S100A8/S100A9 (green) antibodies. Arrows indicate the vesicles containing only hS100A9 or hS100A8. (D) Distribution of hS100A8 and hS100A9 proteins in THP-1 cells by Transmission Electron Microscopy. Black and white boxes represent magnification of areas where S100A8 and S100A9 hetero- or homodimers can be observed respectively. Further, black arrows highlight cytoplasmic vesicles or plasma membrane patches where hS100A9 (big dots) and hS100A8 (small dots) proteins co-localize, while red arrows point to vesicles containing only hS100A9 or hS100A8 homodimers.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Cell Culture, Staining, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: THP-1 cells were cultured for 48h in presence or absence of 10 ng/ml TNFα. S100A8 and S100A9 mRNA expression was determined using qRT-PCR and was normalized to β-actin mRNA level. Data indicate mean ± SD of triplicate samples. Differences between treated and untreated groups are significant at ***P<0.001 by unpaired Student’s t test (A). Intracellular S100A8 and S100A9 protein expression was determined using Western blot (B). THP1 cells were treated with TNFα or left untreated. Cells were harvested either after 48 h (C) or after 24 h and 48 h (D); gently homogenized in homogenization buffer and post-nuclear supernatants (PNSs) were prepared. Using the PNSs as starting material, endosome enriched fractions were prepared using a discontinuous sucrose gradient centrifugation. Fractions were analysed by western blotting using the indicated antibodies. F1 is the interphase between 25% sucrose/homogenization buffer and F2 is the interphase between 35%/25% sucrose. (E) Immunofluorescence analysis of S100A9 localization in TNFα activated THP-1 cells; THP-1 cells cultured for 48 h with TNFα were fixed, permeabilized, stained with anti-S100A9 antibody (green) along with different vesicular marker antibodies anti-Rab5, anti-Rab7 and anti-cathepsin D (all of them marked in the red channel) and analyzed by confocal microscopy.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Homogenization, Gradient Centrifugation, Immunofluorescence, Staining, Marker, Confocal Microscopy

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: THP-1 cells were cultured in triplicate either in the presence or absence of 10 ng/ml TNFα and TNFα + IL-10 (10 ng/ml). Culture supernatant was collected after 48h of incubation and S100A8/S100A9 dimer concentration was determined with ELISA following manufacturer’s protocol (A). Percent cell death was determined using LDH assay (B). In some experiments, THP-1 cells were stimulated with TNFα for 48 h in presence of methylamine (30 μM). In other experiments, cells were stimulated 12 h with TNFα without methylamine, followed by 36 h of culture with the drug. Alternatively, THP-1 cells were stimulated with TNFα; after 36 h, brefeldin A (5 μg/ml) was added and cells were cultured for another 12 h. Culture supernatant was collected after completion of incubation and S100A8/A9 heterodimer (C) and S100A9 (D) concentration was determined with ELISA following manufacturer’s protocol. Values are means of triplicates ± SD. Differences between various treatment groups are significant at **P<0.01, ***P<0.001 and ns = not significant by unpaired Student’s t test.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Cell Culture, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay