Anti akt monoclonal antibody

After the indicated treatments, total cell extracts were obtained and lysed by RIPA buffer (KeyGENBioTECH, Nanjing, China). Protein concentrations were determined according to BCA Protein Assay Kit (Beyotime, Shanghai, China). The extracted proteins in the cell lysates were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The primary antibodies were as follows: rabbit anti-EGFR monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-PTEN monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-p-AKT(Ser473) monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-AKT monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-cyclin D1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bcl2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bax monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-CYP2E1 monoclonal antibody (Epitomics, CA, USA) and mouse anti-β-actin monoclonal antibody (BOSTER, Wuhan, China). Secondary antibodies include HRP-Conjugated AffiniPure Goat Anti-rabbit IgG (ZSGB-BIO, Beijing, China). Immunoreactive proteins were visualized using ECL western blotting detection regents (GE Health-care, Buckinghamshire, UK).

Licochalcone A (LA, ≥95%) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Collagen, thrombin, and U46619 were purchased from Chrono-Log (Havertown, PA, USA). FITC-conjugated anti-P-selectin and PAC-1 antibodies were purchased from Biolegend (San Diego, CA, USA). FITC-conjugated Collagen, phorbol-12, 13-dibutyrate (PDBu), luciferase/luciferin, fluorescein sodium, and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma (St. Louis, MO, USA). Fura 2-AM was purchased from Molecular Probe (Eugene, OR, USA). Anti-phospho PLCγ2 (Tyr⁷⁵⁹), anti-PLCγ2, anti-phospho-(Ser) PKC substrate, anti-phospho-p38 MAPK (Ser¹⁸⁰/Tyr¹⁸²), anti-phospho-p44/42 MAPK (ERK1/2; Thr²⁰²/Tyr²⁰⁴), anti-c-Jun N-terminal kinase (JNK), and anti-phospho-Akt (Ser⁴⁷³) polyclonal antibodies and anti-p38 MAPK, anti-p44/42 MAPK, anti-phospho JNK (Thr¹⁸³/Tyr¹⁸⁵), and anti-Akt monoclonal antibodies were purchased from Cell Signaling (Beverly, MA, USA). The pleckstrin (p47) antibody was purchased from GeneTex (Irvine, CA, USA). The Hybond-P polyvinylidene difluoride membrane, an enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system, horseradish peroxidase (HRP)-conjugated donkey antirabbit IgG, and sheep antimouse IgG were purchased from Amersham (Buckinghamshire, UK). LA was dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C until use.