Journal: PloS one

Article Title: A knock-in mouse model for the R120G mutation of αB-crystallin recapitulates human hereditary myopathy and cataracts.

doi: 10.1371/journal.pone.0017671

Figure Lengend Snippet: Figure 4. Interaction of aB-crystallin with vimentin in wild-type and aB-R120G knock-in lenses. A) Twelve-week-old lens protein extracts were immunoprecipitated with anti-vimentin. Two to four lenses were used per experiment. Immunoprecipitates were immunoblotted using antibodies specific for aB-crystallin. Controls for immunoprecipitation included rabbit serum containing no primary antibody (no antibody controls). Whole cell lysates were also analyzed to assess the total amount of vimentin and aB-crystallin in the lysates. aB-crystallin levels were identical in wild- type and heterozygous whole cell lysates (data not shown). Densitometric scans were obtained, and immunoprecipitated aB-crystallin and whole cell lysate vimentin was determined. Bar graphs to the right show the average of two independent experiments. B, C) Immunohistochemical assessment of co-localization of vimentin and aB-crystallin in aB-R120G mutant lenses. Mid-sagittal lens sections (4 mm) were stained with antibodies against vimentin (green) and aB-crystallin (red) and visualized using a confocal microscope. B) Anterior cortical fibers. Top row, wild-type lens. Bottom row, aB- R120G heterozygous lens. Arrows indicate the areas of dysmorphology. C) Equatorial lens epithelium and fibers. Top row, wild-type lens. Bottom row, aB-R120G heterozygous lens. Note that the heterozygous mutant lenses showed more clusters of vimentin and aB-crystallin (arrows). Four wild type and four aB-R120G heterozygous mutant lenses were analyzed. doi:10.1371/journal.pone.0017671.g004

Article Snippet: Mouse lenses were extracted in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Sigma-Aldrich), lysed for 5 min on ice, and then centrifuged for 10 min at 10,0006 g. Supernatants were treated with a polyclonal primary antibody against vimentin (a gift from Dr. Paul Fitzgerald) conjugated to amino-link plus coupling resin (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol.

Techniques: Knock-In, Immunoprecipitation, Immunohistochemical staining, Mutagenesis, Staining, Microscopy