Ab40660

Alterations in the protein expression levels were analyzed by Western blotting as previously described by our group [42 (link)]. In brief, SKBR3 and ZR75 cells (2 × 10⁶ cells) were seeded and treated with DA and BMS-202, individually and in combination for 48 h. Cell lysates were collected, and equal amounts of protein (30 µg) were resolved on 10% SDS PAGE gels and electroblotted onto PVDF membranes, then probed with the following primary antibodies: anti-rabbit Src family (phospho Y418) (Abcam: ab40660), anti-mouse ErbB2 (Abcam: ab16901), anti-rabbit phosphorylated ErbB2 (Abcam: ab53290), anti-mouse E-cadherin (Cell Signaling: 14,472 S), anti-rabbit vimentin (Cell Signaling: 46,173 S), anti-rabbit β-catenin (Cell Signaling: 8480 S), anti-rabbit phosphorylated β-catenin (Cell Signaling: 4176 S), anti-rabbit AKT (Cell Signaling: 9272 S), anti-rabbit phosphorylated AKT (Cell Signaling: 4060 S), anti-rabbit JNK1/2/3 (Abcam: ab179461). Anti-rabbit GAPDH (Cell Signaling: 8480 S) was used to ensure equal loading of protein samples.
Immunoreactivity was analyzed using the ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer, and blots were imaged using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Quantification was done using ImageJ software as previously described by our group [43 ].

Total cellular protein from the A549 cell lines, NSCLC, and peritumoral tissues was extracted using RIPA buffer and quantified using the BCA assay. Sixty micrograms of protein from each sample was resolved by 10% SDS-PAGE, transferred to PVDF membranes (Millipore), which were then blocked in 5% skim milk in TBST, and incubated with primary antibody overnight at 4°C. The primary antibodies used were as follows: QKI (A7043, ABclonal), epidermal growth factor receptor (EGFR) (ab52894, ABclonal), AGR2 (1A8A8, Proteintech), E-cadherin (#14472, CST), N-cadherin (#13116, CST), p-SRC Y418 (ab40660, Abcam), and p-STAT3 Y705 (ab76315, Abcam). After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Protein bands were visualized using a Tanon 5200 chemiluminescent system (Shanghai, China).