Anti pak4 antibody

Transcriptional activity was analyzed using a 96-well TF Protein Interaction Plate Array I kit (Signosis; FA-3001). Briefly, 10 μg nuclei extracts from ECs were isolated with a nuclear extraction kit (Signosis; SK-0001) and mixed with TF Probe mix I and anti-PAK4 antibody (Cell Signaling Technology; 3242). The mixture was analyzed according to the manufacturer’s instructions. The activity of each transcription factor was normalized as the fold of GATA’s activity.

Total cell lysates were prepared as described in NP-40 buffer (1% NP-40, 150mM NaCl, 2mM EDTA, 50mM Tris-HCl, pH8.0). For the FLAG IP, proteins were incubated with anti-FLAG affinity gel (Sigma) for 1hour at 4°C with rotating. Beads were washed 4 times with wash buffer, and then resuspended in protein loading buffer. For the PAK4 IP, lysates were pre-cleared with Protein A Agarose (Invitrogen) for 1 hour at 4°C and then incubated with 10 μg of anti-PAK4 antibody (Cell Signaling), overnight at 4°C. Antigen-Antibody bound Protein A Agarose pellets were washed 4 times with wash buffer, and then resuspended in protein loading buffer. The released proteins were fractionated by Bis-Tris 4%–20% SDS-PAGE (Invitrogen), and then transferred to nitrocellulose membranes. Immunoblotting was performed as described above using antibodies RhoA, PAK4, Flag, or β-actin antibody as described above.