Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Recombinant HRG binds STC2 and modulates phagocytosis of bioparticles. ( A ) Co-immunoprecipitation of HRG but not inactive HRG with STC2. Recombinant proteins (2 µg each) were separated on SDS-PAGE as individual preparations (loading control) or after mixing and immunoprecipitation (IP) using antibodies against STC2, followed by immunoblotting (IB) as indicated. ( B ) Ratio of HRG (active or inactive) band intensities in the STC2 immunoprecipitate normalized to corresponding active and inactive HRG loading controls. Statistical analysis; Student’s t -test. ( C ) Representative microscope images of U937 monocytes without (left) or with treatment with active HRG (right) in the phagocytosis assay. Green cells have engulfed pH-sensitive fluorescent bioparticles. Scale bar; 50 µm. ( D ) Quantification of phagocytosis efficiency in the different treatment conditions. The proportion of positive (green) phagocytotic U937 cells to all cells per field of vision is shown in relation to the positive cells/total cells in the vitD3 differentiated HRG-treated condition (set to 1). Statistical analysis; Tukey’s multiple comparisons test ( D ). *** p < 0.001; **** p < 0.0001.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Recombinant, Immunoprecipitation, SDS Page, Control, Western Blot, Microscopy, Phagocytosis Assay

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Affinity determination of HRG’s binding to STC2 using QCM. ( A ) The immobilization of STC2 on the QCM LNB sensor surface. ( B ) Sensorgram and kinetic analysis showing chip-immobilized STC2 and binding of HRG at three different concentrations: 50, 100 and 200 nM. Black lines: experimental curves. Red lines: fitted curves. For representative sensorgram shown, three injections per concentration, two independent experiments. ( C ) Sensorgram showing chip-immobilized STC2 and lack of binding of inactive HRG tested at three different concentrations: 50, 100 and 200 nM. For representative sensorgram shown, three injections per concentration, two independent experiments. ( D ) Sensorgram showing chip-immobilized HRG and lack of binding of STC2, tested at three different concentrations: 200 nM, 450 nM and 900 nM. For representative sensorgram shown, two injections per concentration, three independent experiments.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Binding Assay, Concentration Assay

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Binding of HRG and STC2 individually and together to live U937 cells. ( A ) Schematic outline of the experimental setup. Undifferentiated or vitD3 differentiated U937 cells, immobilized on QCM LNB chips with HRG, STC2 or a mix of the two, injected over chip surfaces. ( B ) Real-time qPCR data of CD14 expression normalized to GAPDH on undifferentiated and vitD3 differentiated U937 cells seeded on the QCM chip. Three independent analyses. ( C – F ) Representative sensorgram showing frequency response from injections over undifferentiated ( C , E , G ) and vitD3 differentiated ( D , F , H ) live U937 cells. Black lines: experimental curves. Red lines: fitted curves of the 1:1 interaction model. For representative sensorgrams shown, three injections per concentration, two independent experiments.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Binding Assay, Injection, Expressing, Concentration Assay

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Affinity of HRG for binding to fixed U937 cells. ( A ) Sensorgram showing frequency response to inactive HRG over vitD3 differentiated, fixed U937 cells. ( B ) Sensorgram showing frequency response to four concentrations (125 nM, 250 nM, 500 nM, 1 µM) of STC2 over fixed, undifferentiated (dashed lines) and vitD3 differentiated (straight lines) U937 cells. The mean of two injections is shown. ( C , D ) Sensorgram and kinetic analysis show the binding of HRG at three concentrations (25, 50 and 100 nM) to undifferentiated ( C ) or vitD3 differentiated ( D ), fixed U937 cells. Black lines: experimental curves. Red lines: fitted curves of the 1:1 interaction model. For representative sensorgrams shown, three injections per concentration, three independent experiments. ( E , F ) Sensorgram and kinetic analysis showing frequency response to three concentrations of HRG (25, 50 and 100 nM) to fixed, undifferentiated ( E ) or vitD3 differentiated ( F ) U937 cells after treatment with heparinase. Black lines: experimental curves. Red lines: fitted curves of 1:2 interaction model. For representative sensorgrams shown, three injections per concentration, three independent experiments.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Binding Assay, Concentration Assay