Anti pak1

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.

Anti-PAK1, anti-pStat3 (Y705), and anti-JAK2 antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-p65, anti-Stat3, anti-β-actin, and anti-Lamin b were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-CD44-FITC and anti-CD24-PE antibodies were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Antibodies for ChIP and EMSA experiments were Anti-PAK1 and anti-pStat3. Human PAK1 siRNA and scrambled siRNA were purchased from Bioneer (Daejeon, Korea). The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Antibodies used for western blot included anti Pak1, phospho-Pak1 (Ser199/204), CaMKII and phospho-CaMKII Thr287 pan-antibodies, myc-tag, cleaved caspase-3, PCNA and GAPDH from Cell Signaling Technology (Boston, MA. United States). Anti-GFP was from Santa Cruz Biotechnology (Dallas, TX. United States), and Anti-phospho-Threonine Antibody, clone 20H6.1, was from Sigma-Aldrich (St. Louis, MO. United States).

Freshly sampled human tissues, mouse tissues or cells were flushed with ice-cold PBS and fixed by incubation in 4% paraformaldehyde in PBS overnight at 4 °C. Fixed samples were dehydrated, embedded in paraffin, and sectioned. The sections were de-waxed and rehydrated. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Antigen retrieval was performed by boiling for 15 min in citrate buffer pH 6.0 or Tris-EDTA pH 9.0. Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology). Staining was performed using the Zsbio kit (Beijing, China) according to the manufacturer's instructions.

The 24ST1NLESG cells were transfected by electroporation using the Neon Transfection System (Life Technologies, Carlsbad, CA, USA). Electroporation parameters were 1350 Pulse Voltage (V), 10 Pulse Width (ms), 3 Pulse Number, 2 × 10⁷ cell/mL. Final concentrations were 1 × 10⁷ cell/mL, 1 µM siRNA (siPAK1, Dharmacon, cat # L-003521-00-0020; siPAK2, Dharmacon, cat # L-003597-00-0020; siPAK4, Dharmacon, cat # L-003615-00-0020; siMAPK1, Thermofisher cat # 4390824; siPKA, Thermofisher cat # 4390825; Non-targeting Control, Dharmacon, cat # D-001810-10-20). The efficiency of siRNA knockdown was assessed by Western blot analysis. Anti-PAK1 (cat # 2602S), anti-PAK2 (cat #2608S), anti-PAK4 (cat # 62690), anti-PKA (cat # 4782), anti-MAPK (cat # 9108), and anti-GAPDH (cat # 97166S) antibodies were purchased from Cell Signaling. The efficiency of siRNA knockdown was quantified using ImageJ.