Horseradish peroxidase linked anti rabbit igg

H1299 cells were plated in RPMI media containing 10% FBS at a density of 5.0 × 10⁴/mL into each 100 × 20 mm tissue culture dish. The cells were incubated (37°C/5% CO₂) overnight to allow them to adhere to the plates. The next day, the media were removed and replaced with experimental media containing PCAIs (0 – 5 μM) or 5 μM NSL-100 and then incubated for 24 h. The cells were washed with PBS, lysed with RIPA buffer and the amount of protein in lysates was determined using Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL). Lysate volumes containing 50 μg of protein were boiled in Laemmli sample buffer and subjected to SDS-PAGE. Proteins were then transferred onto PVDF membrane and immunoblotted using antibodies against Rac1, Cdc42 and RhoA (67B9) that were purchased from Cell Signaling Technology (Danvers, MA). Bound antibodies were visualized using horseradish peroxidase-linked anti-rabbit IgG (Santa-Cruz Biotechnology, sc-2004) and ECL reagents (Bio-Rad) according to the manufacturer's recommendation.

H1299 cells that were transfected with YFP-RhoA or the YFP-vector (pZsYellow1-C1) were lysed in RIPA buffer, boiled in Laemmli sample buffer and subjected to SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and immunoblotted using an antibody against pZsYellow (Living Colors Anti-RCFP polyclonal pan antibody) from Clontech (Mountain View, CA). Bound antibodies were visualized using horseradish peroxidase-linked anti-rabbit IgG (Santa-Cruz Biotechnology, sc-2004) and ECL reagents (Bio-Rad) per manufacturer's recommendation.