Anti chicken hrp

For detection of gPrestin expressed in Sf9 cells, samples were separated on 4–20% gradient Mini-Protein precast gels (BioRad), transferred to 0.45 μm nitrocellulose membranes (BioRad) and probed with 1:5000 dilutions of anti-V5 antibodies (Invitrogen) followed by 1:5000 dilutions of goat anti-mouse-HRP antibodies (Jackson Laboratory). For detection of gPrestin expressed in HEK293T cells, anti-GFP (Rockland) and anti-chicken-HRP (Jackson Laboratory) antibodies were used.

Western blotting was performed according to standard procedures. Primary antibodies used were mouse anti-V5 (1:5,000; R960-25; Invitrogen), rabbit anti-Flag (1:2,000; F7425; Sigma-Aldrich), mouse anti–histone-H1 (1:10,000; 05-629; EMD Millipore), mouse antitubulin (1:10,000; T9026; Sigma-Aldrich), chicken anti-HA (1:2,000 and 1:5,000; ET-HA100; Aves Lab), mouse anti-HA (12CA5; 1:2,000; 11 583 816 001; Roche), mouse anti-Dorsal (7A4; 1:500; Developmental Studies Hybridoma Bank), mouse anti-Cactus (3H1; 1:500; Developmental Studies Hybridoma Bank), and rabbit anti-Dif (1:500; a gift from D. Ferrandon, University of Strasbourg, Strasbourg, France). Secondary antibodies used were anti-mouse HRP (1:5,000; PI-2000; Vector Laboratories), anti-rabbit HRP (1:5,000; PI-1000; Vector Laboratories), and anti-chicken HRP (1:10,000; 703-035-155; Jackson ImmunoResearch Laboratories, Inc.).