Anti 3 mst

Western blot assay was carried out as previously described (Wang et al., 2019 (link)). The primary antibodies, including anti-CBS, anti-CSE, anti-3-MST, anti-cyclin E1, anti-cyclin D1, anti-cyclin-dependent kinase-2 (CDK2), anti-cyclin-dependent kinase-4 (CDK4), anti-p27, anti-p21, anti-beclin-1, anti-LC3A/B, anti-P62, anti-phosphatidylinositol 3-kinase (PI3K), anti-AKT, anti-mammalian target of rapamycin (mTOR), anti-phospho (p)-PI3K (Tyr199/Tyr458), anti-p-AKT (Ser473), and anti-p-mTOR (Ser2448) were purchased from Cell Signaling Technology (CST, Danvers, MA, United States). The primary antibodies, including anti-cleaved caspase (cas)-3, anti-cleaved cas-9, anti-cleaved poly adenosine diphosphate-ribose polymerase (PARP), anti-β-actin, and the horseradish peroxidase-conjugated secondary antibody were purchased from Proteintech (Chicago, IL, United States). The immunoreactive bands were visualized by a chemiluminescence detection system (Thermo Fisher, Waltham, MA, United States).

After treatment for 24 h, total protein was extracted from TPC-1, TT, and ARO cells. Western blotting was performed to detect the expression of target proteins. The primary antibodies, including anti-β-actin, anti-CSE, anti-CBS, anti-3-MST, antisulfide-quinone reductase (SQR), antithiosulfate sulfurtransferase (TST), antiphosphatidylinositol 3-kinase (PI3K), anti-phospho (p)-PI3K (Tyr458/Tyr199), anti-AKT, anti-p-AKT (Ser473), antimammalian target of rapamycin (mTOR), anti-p-mTOR (Ser2448), anti-H-RAS, anti-RAF, anti-p-c-RAF (Ser259), anti-MEK1/2, anti-p-MEK1/2 (Ser217/221), antiextracellular signal-regulated protein kinase 1/2 (ERK1/2), anti-p-ERK1/2 (Thr202/Tyr204), and the horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). The results were normalized to the level of β-actin. The reaction was visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). The bands were semiquantified with ImageJ software.