Phosphatase inhibitor cocktail p5726

Tissue samples and platelets for PKC activity assay were prepared. The hippocampus and platelet pellet were homogenized in lysis buffer containing Tris-HCl (20 mM; pH 7.4), EGTA (2 mM), EDTA (5 mM), 0.2% Triton X-100, dithiothreitol (1 mM), protease inhibitor cocktail (P8340), and phosphatase inhibitor cocktail (P5726) (Sigma-Aldrich). Homogenates were centrifuged at 14,000 g for 10 min to remove debris. The concentration of protein was determined using the QuantiPro BCA assay kit (Sigma-Aldrich).
PKC activity was measured using a kit according to the manufacturer’s instructions (ADI-EKS-420A, Enzo Life Sciences), which is based on a solid-phase enzyme-linked immunosorbent assay. It utilizes a specific synthetic peptide as a substrate for subtypes of PKCs and a polyclonal antibody for the phosphorylated form of the substrate. The absorbance was determined at a wavelength of 450 nm, and PKC activity was expressed as a relative activity. Data were normalized to total protein content (μg), as measured by the BCA assay.

Bovine serum albumin (BSA) A7906, progesterone, Coomassie brilliant blue-G, sennoside A, TRITC-labeled phalloidin, fluorescein isothiocyanate (FITC)-labeled PNA, phosphatase inhibitor cocktail P5726, and protease inhibitor cocktail P8340 were purchased from Sigma-Aldrich. Eosin Y was purchased from Biopack. Membrane-permeable Exo enzyme C3 Transferase (C4) was obtained from Cytoskeleton. CAS 1177865-17-6 was obtained from Merck. Cyclosporin A and OA were obtained from Cayman Chemical. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP anti-mouse IgG from Vector Laboratories. PF-3758309 from Medkoo Biosciences. Antibody anti-rabbit IgG-Alexa Fluor 568 from Invitrogen, Thermo Fisher Scientific; while anti-14-3-3 (pan 14-3-3 b8) from Santa Cruz Biotechnology. Anti-COFILIN, anti-phospho-SSH1 (pSSH1; Ser978), anti-phospho-LIMK1/2 (pLIMK1/2; Thr508), and anti-phospho-COFILIN (pCOFILIN; Ser3) antibodies were purchased from Cell Signaling. Anti-PAK4 from Proteintech. Anti-β-tubulin E7 was obtained from Developmental Studies Hybridoma Bank University of Iowa. PF-3758309, cyclosporine A, OA, and sennoside A were dissolved in DMSO; C4, CAS 1177865-17-6, and Eosin Y were dissolved in hexa-distilled water, and phalloidin-TRITC and PNA-FITC were dissolved in PBS.

Protein was extracted using radio immune precipitation (RIPA) lysis buffer complemented with a Protease inhibitor cocktail (Set V, Calbiochem, Dorset, UK) and a phosphatase inhibitor cocktail (P5726 (Sigma, Dorset, UK)). Cell lysates were vortexed every 2 min and kept on ice for 10 min before being centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatants were collected and total proteins were quantified using a Modified Lowry assay (Thermo Scientific, Oxford, UK). All experiments were done in 3 biological and experimental repeats.