Enhanced chemiluminescence western blotting detection reagents

Western blotting of Gi proteins was performed using specific antibodies, as described previously.20 After SDS‐PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose membrane with a semi–dry transfer blot apparatus (Bio‐Rad Laboratories) at 15 V for 45 minutes. After transfer, the membranes were washed twice in PBS and were incubated in PBS containing 5% skim milk at room temperature for 1 hour. The blots were then incubated with the following specific antibodies: Giα‐2 (L5), Giα‐3 (C‐10), and dynein (74‐1; Santa Cruz Biotechnology) incubated in PBS containing 0.1% Tween 20 overnight at 4°C. The antigen–antibody complexes were detected by incubating the blots with goat anti–rabbit immunoglobulin G (Bio‐Rad Laboratories) conjugated with horseradish peroxidase for 1 hour at room temperature. The blots were then washed 3 times with PBS before reacting with enhanced chemiluminescence Western blotting detection reagents (Santa Cruz Biotechnology). Quantitative analysis of the protein was performed by densitometric scanning of the autoradiographs using the enhanced laser densitometer LKB Ultroscan XL and quantified using the Gelscan XL evaluation software (version 2.1) from Pharmacia.

After membrane protein extraction as described above, equal amounts of cellular proteins were mixed with 5x sample buffer and heated at 100℃ for 5 min. Proteins were then resolved on an 8% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred onto an ECL nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) using Tris buffer [0.025 M Tris-HCI (pH 6.8), 0.192 M glycine, and 20% MeOH]. The membrane was blocked for 1 h at room temperature with 5% skim milk in TBS-Tween 20, incubated overnight at 4℃ with anti-CD47 antibody (BD Bioscience) and anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and then incubated with horseradish peroxidase-conjugated antirat secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Finally, the membrane was developed using enhanced chemiluminescence Western blotting detection reagents (Santa Cruz Biotechnology) and quantified via densitometry.