Anti p akt

Cells were harvested and lysed with modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM sodium chloride, 1 mM ethylenediamine tetra-acetic acid, 1% NP40, 0.25% sodium deoxycholate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). Whole-cell lysates were subjected to SDS-PAGE, transferred to a PVDF membrane (Immobilon®-P), and probed with primary antibodies for the target proteins at 4°C overnight. The primary antibodies used included anti-p84 (#GTX70220, GeneTex), anti-p-Akt (#GTX121937, GeneTex), anti-p-ERK [24 (link)] Cell Signaling, anti-p-p38 (#9211, Cell Signaling), anti-p-JNK (#4668, Cell Signaling), and anti-CEBPD (#GTX115047, GeneTex). The specific proteins were detected by incubation with a peroxidase-conjugated secondary antibody at room temperature for 1.5 h. Proteins were visualized using an ECL kit (PerkinElmer).

The spheroids were formed by plating 1 × 10⁶ cells per well in serum-free media with supplementation of growth factors and treated with various concentrations of aqueous extractions of BJ (0, 5, 10, and 15 mg/mL) for 12 h. The harvested cell pellets were lysed and the protein concentrations determined using a Bio-Rad protein assay kit for (Hercules, CA, USA) before being resolved by electrophoresis and transferred to nitrocellulose membrane. The blots were blocked with 5% non-fat milk and incubated with 1:2000 dilutions of primary antibodies, including anti-pAkt (GTX128414); anti-PARP (GTX112864); anti-EGFR (GTX121919); anti-pEGFR (GTX61507), anti-ABCG2 (GTX100437), anti-Nanog (GTX100863), anti-CD133 (GTX100567), anti-Sox2 (GTX627405), and anti-ALDH1A1 (GTX123973), from GeneTex. Membranes were then incubated with 0.3 µg/mL of peroxidase-conjugate anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific) and detected with enhanced chemiluminescence substrate (Thermo Fisher Scientific). The loading control was incubated with anti-GAPDH antibody (GTX100118, GeneTex). The signals were visualized with enhanced LAS-4000 (FUJIFILM) apparatus and the band intensities of images analyzed using ImageJ software.