Journal: Molecular Biology of the Cell

Article Title: The Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for dendrite formation in melanocytes

doi: 10.1091/mbc.E11-04-0324

Figure Lengend Snippet: Rab21 and Rab32/38 are present in distinct membrane compartments in melanocytes. Immunoaffinity purification of Rab32-containing vesicles/organelles from crude membrane fractions of B16-F1 cells was performed by using anti-Rab32 IgG (lane 3) or control rabbit IgG (lane 2) as described in Materials and Methods . The Rab32-containing membrane compartment was analyzed by 10% SDS–PAGE, followed by immunoblotting with anti-Varp antibody (0.4 μg/ml), anti-tyrosinase antibody (0.3 μg/ml), anti-Rab32 antibody (1.5 μg/ml), anti-Rab38 antibody (0.9 μg/ml), anti-Rab21 antibody (0.2 μg/ml), anti-EEA1 antibody (1/1000 dilution), and anti-GM130 antibody (0.3 μg/ml). Note that Rab21 was not copurified with the Rab32-membrane compartment, which clearly includes Rab38, tyrosinase, and Varp but not EEA1 or GM130. Input means 1% volume of the crude membrane fractions used for immunoaffinity purification. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

Article Snippet: Anti-EEA1 mouse monoclonal antibody and anti-GM130 mouse monoclonal antibody were from Cell Signaling Technology (Danvers, MA) and BD Biosciences (San Jose, CA), respectively.

Techniques: Membrane, Immunoaffinity Purification, Control, SDS Page, Western Blot