Rabbit anti p pak4 ser474

Cytoplasmic and nuclear extracts from RL95-2 cells were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce; Thermo Scientific, Rockford, IL, USA). Primary antibodies were the following: rabbit anti-Pak4 (1:1000, Abcam, Cambridge, UK), rabbit anti-p-Pak4 Ser⁴⁷⁴ (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:2000, ProteinTech Group, Chicago, IL), rabbit anti-tubulin (1:1000, Cell Signaling Technology), and rabbit anti-histone H3 (1:1000, Cell Signaling Technology).

Cells were lysed using ProteoJET Mammalian Cell Lysis Reagent (MBI Fermentas, ON, Canada) with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Total protein concentration was estimated using the BCA method (Pierce, Rockford, IL, USA). A total of 60 μg of protein was separated on an 8% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated with primary antibodies. Signal was detected using the BeyoECL Plus (Beyotime, Shanghai, PR China).
Primary antibodies were: rabbit anti-Pak4 (1:1000, Abcam, Cambridge, UK), rabbit anti-p-Pak4 Ser⁴⁷⁴ (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:2000, ProteinTech Group, Chicago, IL), rabbit anti-AKT1 (1:1000, Epitomics, Burlingame, CA, USA), and rabbit anti-p-AKT Ser⁴⁷³ (1:1000, Epitomics, Burlingame, CA, USA).