Anti pak2

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.

For total protein extraction, the tissue and cell samples were lysed in Radio Immunoprecipitation assay (RIPA) lysis solution (CWBIO) with protease and phosphatase inhibitors (Roche, Madison, WI, USA), followed by denaturation at 95 °C for 10 min. The denatured supernatant was separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (12%) and transferred to polyvinylidene difluoride membranes at 4 °C for 90 min. The membranes were blocked with BSA (5%) or defatted milk (5%) and incubated with primary antibody in BSA (5%) at 4 °C overnight. The primary antibodies were obtained from Cell Signaling Technology, including anti‐PAK2 (1:1000; Cat^# 2608), anti‐p‐Smad1/5/9 (D5B10) (1:1000; Cat^# 13820), anti‐p‐Smad1/5(41D10) (1:1000; Cat^# 9516), and anti‐p‐PAK1 (Ser144)/PAK2 (Ser141) (1:1000; Cat^# 2606); and from Abcam, including Smad1/5/9 (1:1000; Cat^# ab66737). The secondary antibodies used were anti‐rabbit IgG conjugated to horse radish peroxidase (HRP) (Cat^# 7074, Cell Signaling Technology) and anti‐mouse IgG conjugated to HRP (Cat^# 7076, Cell Signaling Technology). Signals were detected using the FluorChem E imaging system (Cell Biosciences, San Jose, CA, USA) and analyzed using ImageJ software (version 1.4.3.67). The relative levels of these proteins were normalized to that of GAPDH (1:5000; Cat^# 60004, Proteintech, Rosemont, IL, USA).

Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The protein containing PVDF membranes were probed with different primary antibodies: anti-PAK2 (2615, Cell Signaling Technology, Beverly, MA, USA), anti-c-Myc (9402, Cell Signaling Technology, Beverly, MA, USA), anti-CCND1 (ab134175, Abcam, Melbourne Australia), anti-PKM2 (4053, Cell Signaling Technology, Beverly, MA, USA), anti-Active β-Catenin (05665, Millipore, Burlington, USA), anti-GAPDH (5174, Cell Signaling Technology, Beverly, MA, USA), anti-β-Catenin (9562, Cell Signaling Technology, Beverly, MA, USA) and anti-flag (NBP1-06712SS, Novus Biologicals, Littleton, CO, USA) in a 1:1000 dilution. After 2 h of primary antibody incubation at room temperature, membranes were washed with 1× TBST (tris-buffered saline and Tween-20) and incubated with secondary antibodies Alexa-Flour 680 anti-rabbit IgG (A21109, Thermo Fisher Scientific, Waltham, MA, USA) and Alexa-Flour 790 goat anti-mouse IgG (A28182, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. The membrane was washed, and bands were visualized using an Odyssey membrane Scanning system (Li-Cor Biosciences, Bad Homburg, Germany).

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.

Total protein was extracted from H9c2 cells or mouse left ventricle tissue by the radio immunoprecipitation assay (RIPA) Lysis Buffer (Solarbio, Beijing, China) according to the manufacturer’s instructions. Proteins were separated by electrophoresis on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10–12% polyacrylamide gels) and then transferred to polyvinylidene fluoride (PVDF) membranes. Subsequently, the PVDF membranes were blocked in 5% non-fat milk for 2 h and then incubated overnight at 4°C with anti-PAK2 (1:1,000, Cell Signaling Technology, Danvers, MA, United States), or anti-XBP1s (1:1,000, Cell Signaling Technology, Danvers, MA, United States), or anti-β-actin (1:2,000, Santa Cruz Biotechnology, Dallas, TX, United States), or anti-GAPDH (1:100,000, ABclonal, Wuhan, China), or anti-cas-12 (1:2,000, Abcam, United States) primary antibodies after washing with TBS-Tween 20 (TBST) three times, 10 min each time. Horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated at room temperature for 1 h, then washed with TBST three times, 10 min each time. Membranes were visualized using enhanced chemiluminescence. Protein expression was quantified using ImageJ, and β-actin or GAPDH was used as the internal control.