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: Figure 1. 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 im- munoprecipitation (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) [24], 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