Anti pak5

Anti-PAK1, anti-PAK2 and anti-GEFH-1 were purchased from Cell Signalling Technology. Anti-c-Myc and anti-PAK5 were acquired from Santa Cruz. Anti-Cortactin from Upstate. Anti-GAPDH from Millipore. Anti-p-GEFH-1 (Ser⁸⁸⁵) from Abcam. Anti-PAK3 from New England Biolabs and anti-PAK6 from Calbiochem. Anti-GFP from Roche Life Science. Anti-β-Actin and anti-β-Tubulin from Sigma Aldrich. Anti-HMWMAA and anti-human IgG kind gift from Sophia Karagiannis, King's College London (KCL). Anti-PAK4 was previously described [17 (link)]. Horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from DAKO. The Alexa Fluor 488 conjugated antibodies and Phalloidin from Invitrogen. GFP-PAK1r, GFP-PAK4r, HA-PAK4r and HA-PAK4K350/351Mr were constructed by site-directed mutagenesis, according to the manufacturer's instructions, using the QuikChange Multisuite II kit (Stratagene). The Myc-PDZ-RhoGEF and Myc-PDZ-RhoGEFΔDH were kind gifts from John Masters, University College London (UCL). The RhoA Biosensor was generously provided by Maddy Parsons, (KCL). IPA-3 was purchased from Sigma.

Cells were lysed in IP lysed buffer (KeyGen BioTECH) with cocktail of protease/phosphatase inhibitors (1:100, Sigma Aldrich, MO, USA), and then added anti-PAK5 (1:100, Santa Cruz, USA) and Mouse IgG (Beyotime) incubation overnight at 4 °C. 40 μl Protein A/G beads (Santa Cruz) were used for binding to antibodies. Wash beads and heat with 2 × loading buffer. Immunoprecipitated proteins were examined by Western Blot.