Journal: Kidney international

Article Title: Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis.

doi: 10.1038/ki.2008.19

Figure Lengend Snippet: Figure 1 | Characterization of antibodies. (a) Detection of immunoprecipitated 3xFLAG-tagged nephrin transiently expressed in COS cells. 3xFLAG-tagged nephrin immunoprecipitated with anti-FLAG M2 antibody was run on SDS–PAGE. With or without pervanadate treatment (100 mM for 30 min), anti-FLAG M2 antibody detected nephrin. However, both anti-pY1208 and anti-pY1228 antibodies detected a band only in the immunoprecipitated nephrin from pervanadate-treated cells. (b) Immunoblot of glomerular lysates with anti-pY1208 and anti-pY1228 antibodies. Lanes 1 and 2 are lysates from glomeruli before and after (4 days) puromycin treatment, respectively. Signals detected without the antigen peptide completely disappeared after immunoabsorption of the antibody with the corresponding phosphorylated peptide (10 mg ml1), but not with the non-phosphorylated peptide. (c) Immunoblot of glomerular lysates with anti-podocin antibody. A single band around 40 kDa was detected with the anti-podocin antibody, which was immunoabsorbed with the antigen peptide (10 mg ml1).

Article Snippet: After blocking nonspecific binding with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20, the blots were incubated with a 1:100 dilution of anti-nephrin antibody (Santa Cruz Technology), antipodocin antibody, or anti-phosphorylated nephrin antibodies.

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: Kidney international

Article Title: Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis.

doi: 10.1038/ki.2008.19

Figure Lengend Snippet: Figure 4 | Immunofluorescence of phosphorylated nephrin, total nephrin, and podocin in glomeruli of rats with PAN nephrosis. Glomeruli in PAN nephrosis on days 0, 4, 7, and 28 were stained with antibodies to phosphorylated nephrin (pY1208 (a) and pY1228 (b)), total nephrin (c), and podocin (d).

Article Snippet: After blocking nonspecific binding with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20, the blots were incubated with a 1:100 dilution of anti-nephrin antibody (Santa Cruz Technology), antipodocin antibody, or anti-phosphorylated nephrin antibodies.

Techniques: Immunofluorescence, Staining

Journal: Kidney international

Article Title: Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis.

doi: 10.1038/ki.2008.19

Figure Lengend Snippet: Figure 6 | Immunofluorescence of phosphorylated nephrin, total nephrin, and podocin in glomeruli of human normal kidneys and minimal change nephrosis. Biopsy specimens from donor kidneys and minimal change nephrosis were immunostained with anti-pY1228 (a), anti-total nephrin (b), and anti-podocin (c) antibodies. Three representative cases were shown. Signals were detected by using LSM510-Meta confocal microscopy under a constant laser and detector setting.

Article Snippet: After blocking nonspecific binding with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20, the blots were incubated with a 1:100 dilution of anti-nephrin antibody (Santa Cruz Technology), antipodocin antibody, or anti-phosphorylated nephrin antibodies.

Techniques: Immunofluorescence, Confocal Microscopy

Journal:

Article Title: Involvement of Sialoadhesin in Entry of Porcine Reproductive and Respiratory Syndrome Virus into Porcine Alveolar Macrophages

doi: 10.1128/JVI.77.15.8207-8215.2003

Figure Lengend Snippet: Porcine sialoadhesin mediates endocytosis of MAb41D3. Alveolar macrophages were incubated for 1 h at 37°C with MAb41D3 (A) or with control MAb MIL-2 anti-CD14 (B). After fixation, washings, and permeabilization, cells were incubated with FITC-conjugated goat polyclonal anti-mouse immunoglobulins and analyzed by confocal microscopy. (C) As a control, cells were fixed, incubated for 1 h at 37°C with MAb41D3, washed, permeabilized, and incubated with the same conjugated polyclonal antiserum as above. (D) Macrophages were incubated at 4°C with MAb41D3 before being shifted to 37°C to allow endocytosis. At different time points (0, 30, 60, 90, and 180 min), cells were shifted back to 4°C, stained with FITC-labeled goat anti-mouse antibodies, and analyzed by flow cytometry. Each value reflects the means ± standard deviation (error bars) of three experiments. (E to G) Macrophages were incubated with MAb41D3 and incubated at 37°C for 10 min before fixation and permeabilization. Cells were analyzed by confocal microscopy after performing a double immunofluorescence staining to detect porcine sialoadhesin (E) and clathrin (F). (G) Overlay of panels E and F. Symbols: Arrowheads, colocalization; arrows, no colocalization. Bar: 10 μm.

Article Snippet: Twenty hours postinoculation with the different PRRSV strains, cells were washed with PBS, fixed with methanol, incubated with a 1:20 dilution of antinucleocapsid MAb P3/27 ( 62 ) in PBS containing 10% heat-inactivated goat serum (PBS-G) for 1 h at 37°C, and washed with PBS before incubation with a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat polyclonal anti-mouse immunoglobulins (Molecular Probes) in PBS-G for 1 h at 37°C.

Techniques: Incubation, Confocal Microscopy, Staining, Labeling, Flow Cytometry, Standard Deviation, Double Immunofluorescence Staining