Comprehensive Antibody Validation Protocol

Anti-CDC42 (#10155-1-AP, 1:1000 for WB), anti-PAK4 (#14685-1-AP, 1:1000 for WB), anti-PAK1(#21401-1-AP, 1:1000 for WB), monoclonal anti-GST(#66001-2-Ig, 1:10000 for WB), polyclonal anti-HA (#51064-2-AP, 1:5000 for WB), monoclonal anti-SIRT2 (#66410-1-IG, 1:10000 for WB), anti-beta tubulin (#10094-1-AP, 1:2000 for WB), MMP-2 (#10373-2-AP, 1:1000 for WB), MMP-9 (#10375-2-AP, 1:1000 for WB) were purchased from Proteintech; Monoclonal anti-HA (#AB0025, 1:5000 for WB); E-cadherin (#BS1098, 1:1000 for WB) were from Bioworld; Monoclonal anti-CDC42 (#sc-8401, 2 μg per 500 μg of total protein for Immunoprecipitation), anti-p-PAK4 (sc-135775, 1:200 for WB) were from Santa Cruz; Anti-p-PAK1 (#2601, 1:1000 for WB), anti-p-AKT (#4060, 1:1000 for WB), anti-AKT (#4691, 1:1000 for WB), anti-p-ERK (#9101, 1:1000 for WB), anti-ERK (#4370, 1:1000 for WB), anti-p-p38 (#9211, 1:1000 for WB), anti-p38 (#9212, 1:1000 for WB), anti-p-JNK (#9251, 1:1000 for WB), anti-ERK (#9252, 1:1000 for WB), anti-caspase 3 (#14220, 1:1000 for WB) and anti-PARP (#9542, 1:1000 for WB) were from Cell Signaling Technology; Monoclonal anti-HA (#M180, 1:500 for Immunoprecipitation), monoclonal anti-Flag (#M185, 1:500 for Immunoprecipitation), polyclonal anti-Flag (#PM020, 1:1000 for WB) and deacetylase inhibitors TSA (#9950) were from MBL; Deacetylase inhibitor NAM (#N1651) was from APExBIO; CBP/p300 inhibitor A-485 (#N1651) was from MCE; SIRT2 inhibitor AK7 (#4754–10) was from Tocris Bioscience; Staurosporine (#abs810006) was from Absin; Polybrene (#H9268) and puromycin (#P8833) were from Sigma-Aldrich. The anti-CDC42 K153Ac specific polyclonal antibody was raised against a synthetic peptide (Jiaxuan Biotech). In brief, the immune peptide DLKAVK(Ac)YVECSALTQKGL was used as antigen to immunize rabbits. During 45 days, rabbits were immunized for four times, and the antiserum was collected, and control peptide DLKAVKYVECSALTQKGL was used to remove non-specific antibody. The sensitivity and specificity of antibody were evaluated by ELISA and WB.