Anti np protein

After drug treatment, the cell lysate was separated by SDS-PAGE and transferred to nitrocellulose membrane. After being blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (v/v) and 5% BSA (w/v) at room temperature for 2 h, the membranes were rinsed and incubated at 4 °C overnight with anti-NP protein (Santa Cruz, USA), anti-phosphorylated PI3K, Akt, ERK1/2, and NF-κB antibodies, or anti-GAPDH, β-actin, and α-tubulin antibodies (Cell Signaling Technology, Danvers, USA) as control. The membranes were washed and incubated with AP-labeled secondary antibody (1:2000 dilutions) at RT for 2 h. The protein bands were then visualized by incubating with the developing solution (p-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP) at RT for 30 min. The relative densities of proteins were all determined by using ImageJ (NIH) v.1.33 u (USA).

After drug treatment, the cell lysate was separated by SDS-PAGE and transferred to nitrocellulose membrane. After being blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (v/v) and 5% BSA (w/v) at room temperature for 2 h, the membranes were rinsed and incubated at 4 °C overnight with anti-NP protein (Santa Cruz, USA), anti-phosphorylated NF-κB, Akt, EGFR, PKCα antibodies, or anti-β-actin and GAPDH antibodies (Cell Signaling Technology, Danvers, USA) as control. The membranes were washed and incubated with AP-labeled secondary antibody (1:2000 dilutions) at RT for 2 h. The protein bands were then visualized by incubating with the developing solution (p-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP)) at RT for 30 min. The relative densities of proteins were all determined by using ImageJ (NIH) v.1.33 u (USA).