Journal: The American Journal of Pathology
Article Title: Differential Expression of D-Type Cyclins in Podocytes in Vitro and in Vivo
Figure Lengend Snippet: A: Western blot analysis for cyclin D1 and cyclin D3 in conditionally immortalized podocytes in vitro . Cyclin D1 was abundant in proliferating podocytes grown under permissive conditions. The levels of cyclin D1 decreased when podocytes were grown under restrictive conditions, and this coincided with a decrease in proliferation, and the development of a differentiated phenotype. There was a progressive increase in cyclin D3 levels when grown under restrictive conditions, compared to permissive conditions. Tubulin was used to assure equal loading. b: Immunofluorescence for cyclin D1 and cyclin D3 in conditionally immortalized podocytes. Cyclin D1 stains positive in proliferating podocytes grown under permissive conditions ( b ), and is barely detected under growth restrictive conditions ( D ). Hoechst staining was used as a nuclear counterstain ( A and C ). Cyclin D3 staining was not detected in proliferating podocytes ( F ), but was abundant in quiescent and differentiated podocytes grown under growth restrictive conditions ( H ). Hoechst staining was used as a nuclear counterstain ( E , G ).
Article Snippet: Indirect immunoperoxidase immunostaining was performed as previously reported using the following primary antibodies incubated overnight at 4°C: cyclin D1 (1:1000, mouse monoclonal, clone DCS-6; Oncogene, Boston, MA), cyclin D1 (1:600, mouse monoclonal, MS-210; Neomarkers, Fremont, CA), cyclin D1 (1:100, mouse monoclonal, clone A-12; Santa Cruz, Santa Cruz, CA), cyclin D3 (1:300, mouse monoclonal, clone DCS-22; Neomarkers), WT-1 (1:1200, rabbit polyclonal, clone C-19; Santa Cruz), Synaptopodin (1:50, gift of Dr. Peter Mundel, Departments of Medicine and Anatomy, Albert Einstein College, Bronx, NY) and Glepp1 (1:50; gift of Dr. Roger Wiggins, Department of Internal Medicine, University of Michigan, Ann Arbor, MI).
Techniques: Western Blot, In Vitro, Immunofluorescence, Staining