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In this study, proteins were extracted using a protein extraction kit (Sangon Biotech), and the bicinchoninic acid (BCA) assay (Sangon Biotech) was used to determine the total protein content. After denaturation for 5 min, total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA) via a constant current flow at 200 mA. Subsequently, the PVDF membranes were incubated with Bcl-2 (1:10,000), Bax (1:2000), Caspase-3 (1:5000), M-CSF (1:1000), RANKL (1:3000), OPG (1:3000), p-p65 (1:1000), p65 (1:1000), p-p38 (1:1000), p38 (1:5000), p-JNK (1:1000), JNK (1:1000), p-AKT (1:2000), AKT (1:2000), p-ERK (1:1000), ERK (1:1000), RANK (1:2000), NF-κB (1:1000), NFATc1 (1:10,000), and c-Fos (1:1000) antibodies (Abcam, Cambridge, MA) for 12 h at 4°C. TBS buffer was used to wash the PVDF membranes, and secondary antibodies (Abcam) were added and incubated at room temperature for 1 h. After the membranes were washed three times, chemiluminescent reagents were added, and the grayscale values of the bands were analyzed using ImageJ software. Each experiment was independently repeated 3 times. GAPDH was used to quantify the expression of various proteins.

NSCLC cells were lysed in RIPA lysis buffer (KeyGEN, Nanjing, China), and the concentration of protein was detected by BCA Assay kit (Solar life science, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was performed to separate the equal amounts of protein (30 μg), and proteins were then shifted onto polyvinylidene difluoride membrane (PVDF, Thermo Fisher Scientific). Five percent nonfat dried milk in TBST was applied to block the PVDF membrane for 1 h. PVDF membrane was incubated at 4°C overnight with the primary antibodies: E-cadherin (Abcam, Cambridge, MA, USA, 1:1000), N-cadherin (Abcam, 1:1000), p-Akt (Abcam, 1:1000), Akt (Abcam, 1:1,000), p-ERK (Abcam, 1:1000), ERK (Abcam, 1:1000), cleaved caspase 3 (Abcam, 1:1000), p27 Kip1 (Abcam, 1:1000), cyclin D1 (Abcam, 1:1000), CDK2 (Abcam, 1:1000) and β-actin (Abcam, 1:1000). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Abcam; 1:5000) for 1 h. Protein bands were visualized using the ECL kit (Thermo Fisher Scientific). β-actin was used as the loading control. IPP 6.0 (Image-Pro Plus 6.0) was used for the densitometry analysis.

Repetitive transcranial magnetic stimulation was purchased from Yingzhi Company (M-100 Ultimate). All reagents are listed as follows: phosphorylated-AKT (p-AKT) (4060, Cell Signaling), AKT (4691, Cell Signaling), phosphorylated extracellular signal-regulated kinase (p-ERK) (50011, Abcam), ERK (184699, Abcam), phosphorylated epidermal growth factor receptor (p-EGFR) (40815, Abcam), EGFR (52894, Abcam), NMDA receptor (NMDAR1) (17345, Abcam), p62 (56416, Abcam), LC3 (192890, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR) (5536, Cell Signaling), mTOR (2983, Cell Signaling), phosphorylated protein kinase A (p-PKA) (32390, Abcam), PKA (75993, Abcam), Oct4 (2750, Cell Signaling), Nanog (8822, Cell Signaling), Sox2 (3579, Cell Signaling), GAPDH (8245, Abcam), dizocilpine maleate (MK801) (15084, MCE), 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U0126) (1901, Beyotime), 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO) (8317, Selleck), Fluo-4/AM (1060, Beyotime), horseradish peroxidase (HRP)-mouse (931, Sigma), HRP-rabbit (934, Sigma), Alexa Fluor 488 (150077, Abcam), Alexa Fluor Cy3 (97035, Abcam), and Cell Counting Kit-8 (CCK-8) (0037, Beyotime).