Anti mammalian target of rapamycin

Radioimmunoprecipitation assay (RIPA) lysate buffer containing protease inhibitors and phosphatase inhibitors was used to lyse LV tissue, and the total protein in each sample was collected and quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Electrophoresis was performed to separate the total protein using 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, and the separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight and then incubated with secondary antibodies at room temperature for 1 h. The primary antibodies included anti-LKB1, anti-p-LKB1, anti-adenosine 5′-monophosphate-activated protein kinase (AMPK), anti-p-AMPK, anti-mammalian target of rapamycin (mTOR), anti-p-mTOR, anti-Bax, anti-Bcl2, anti-cleaved caspase-3, and anti-GAPDH antibodies (all from Cell Signaling Technology). The blots were scanned using a two-color infrared imaging system (Odyssey, USA).

Seventy-two hours post-transfection, total protein was extracted from SH-SY5Y and SK-N-SH cells. Western blotting was performed to detect the expression of target proteins. The primary antibodies, including anti-extracellular signal-regulated protein kinase 1/2 (ERK1/2), anti-phospho (p)-ERK1/2 (Thr202/Tyr204), anti-c-Jun N-terminal kinase (JNK), anti-p-JNK (Thr183/Tyr185), anti-p38, anti-p-p38 (Thr180/Tyr182), anti-phosphatidylinositol 3-kinase (PI3K), anti-p-PI3K (Tyr458/Tyr199), anti-Akt, anti-p-Akt (Ser473), anti-mammalian target of rapamycin (mTOR), and anti-p-mTOR (Ser2448) antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-PCNP, Anti-B-cell lymphoma-2 (Bcl-2), anti-Bcl-2-associated X protein (Bax), anti-B-cell lymphoma-extra large (Bcl-xl), anti-Bcl-xl/Bcl-2-associated death promoter (Bad), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-Cleaved poly adenosine diphosphate-ribose polymerase (PARP), and anti-GAPDH antibodies were purchased from ProteinTech (Chicago, IL, USA). The horseradish peroxidase-conjugated secondary antibody was purchased from Cell Signaling Technology. The results were normalized to the level of GAPDH. The reaction was visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). The bands were semi-quantified with Image J software.

Western blot was performed using the following primary antibodies anti-phospho-Akt-Ser473 (Cell Signaling, Danvers, MA, USA), anti-Akt (Cell Signaling), anti-phospho mTOR- Ser 2448 (Cell Signaling), anti-mammalian target of rapamycin (mTOR; Cell Signaling), IGF-1 (Sigma, Dorset, UK) and α-Tubulin (Sigma) as previously described.^{47 (link),48 (link)} Brightness and contrast were linearly adjusted using power point.