Bound proteins were visualized by autoradiography.
After washing the membranes, the bound proteins were visualized by a luminol detection system (Santa Cruz).
Bound proteins were visualized with the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) and quantified with the Multigauge software (Fuji Film, Tokyo, Japan).
Following incubation with horseradish peroxidase-conjugated secondary antibody (Southern Biotechnology, Birmingham, AL, USA), bound proteins were visualized by chemiluminescence ECL (Amersham Biosciences, Freiburg, Germany).
After incubation with peroxidase-coupled antimouse IgG (Santa Cruz) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using a BioImaging System (UVP Inc., Upland, CA, USA).
(E, F ) HEK293T cells expressing FLAG-Aven and FLAG-AvenΔRGG were processed as in panel C, D except the bound proteins were visualized by immunoblotting with anti-FLAG antibodies.
The antibody-bound proteins were visualized using an ECL detection kit (Amersham Biosciences, Piscataway, NJ, USA) or Alexa Fluor 488 (green) donkey anti-mouse IgG (Invitrogen, La Jolla, CA, USA).
Bands of antibody-bound proteins were visualized by chemiluminescence, the blotted membrane was scanned for densitometry with a PDI imaging scanner (Agfa Japan, Tokyo), and the data were analyzed with NIH Image software (ImageJ for Mac OS, version 1.47).
Bands of antibody-bound proteins were visualized by chemiluminescence; the blotted membrane was scanned for densitometry with a PDI imaging scanner (Agfa Japan, Tokyo), and the data were analyzed with NIH Image software.
Bands of antibody-bound proteins were visualized by chemiluminescence, densitometry of the blotted membrane was done with a PDI imaging scanner (Agfa Japan, Tokyo), and the data were analyzed with NIH Image software.
