Western Blot Analysis of Immunoreactive Proteins

Immunoreactive proteins were detected as previously described [9 (link)]. Briefly, 20 µg of cytosolic and nuclear protein extracts were fractionated in SDS-PAGE gels at 6% for the detection of DOCK9, DOCK10, and DOCK11 and at 10% for the detection of HDAC1 and actin, and electroblotted onto nitrocellulose membranes. Blots were blocked, incubated with primary Abs (Table 2) in TBST with 0.5% skim milk for 2 h, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary Abs—rabbit, mouse or goat immunoglobulins (Igs) (Table 2)—in TBST with 2.5% skim milk for 1 h. After final washes, immunoreactive proteins were detected using the Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) in the Molecular Imager ChemiDoc™ XRS+ with Image Lab software (Bio-Rad Laboratories, Hercules, CA), which provides the tools to measure the intensity of the bands. Abs and their dilutions are listed in Table 2. The primary (1ary) Abs that were obtained from the mouse or rat (Actin, HA) were monoclonal (Mo) and those obtained from the rabbit or goat (DOCK9, DOCK10, DOCK10.1, DOCK11, HDAC1) were polyclonal (Po).