Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs
Figure Lengend Snippet: ( A ) CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with plasma-derived Z-AAT, lipopolysaccharide (LPS), or M-AAT in the concentrations as indicated, or with RPMI medium alone (control). The data from n = 6 independent experiments are presented as median (IQR) in box and whisker plot format; lines represent medians in each box. Measurements were carried out in duplicates. p-Value was calculated by nonparametric Kruskal-Wallis test. ( B ) Representative uncut Western blot (n = 3 independent experiments) of CX3CR1 in RIPA lysates prepared from PBMCs incubated for 18 hr alone or with LPS (1 µg/ml), M-AAT (1 mg/ml), or Z-AAT (0.5 mg/ml). For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. Relative intensities were calculated for each band using the ratio relative to β-actin, as a loading control, and then normalized by the experimental control. ( C ) For analysis of cellular AAT, the same lysates were separated under non-reducing conditions. Western blots were probed with polyclonal rabbit anti-human AAT recognizing monomeric, polymeric, or truncated forms of AAT. One representative blot from n = 3 independent experiments is shown. β-Actin was used for a loading control. ( D ) and ( E ) Co-distribution of Z-AAT polymers with CX3CR1 in human total PBMCs incubated with 0.5 mg/ml Z-AAT polymers for 18 hr. ( D ) Immunofluorescence microscopy revealed co-localization of Z-AAT polymers ( red ) with CX3CR1-positive structures ( green ). Arrows point areas of co-localization. Scale bar, 10 µm. ( E ) Confocal microscopy 3D stack with orthogonal reconstruction shows an aggregate of Z-AAT polymers ( red ) surrounded by CX3CR1-positive ( green ) cellular extensions forming a cap-like structure (arrowhead). Scale bar, 5 µm. The images with indicated channels merged and the corresponding differential interference contrast (DIC) image are presented. 4', 6- Diamidino 2-phenylindole (DAPI) was used for nuclei staining ( blue ). Figure 2—source data 1. Source file, containing original data for , to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).
Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.
Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Derivative Assay, Whisker Assay, Western Blot, SDS Page, Immunofluorescence, Microscopy, Confocal Microscopy, Staining