Cleaved caspase 8 asp391 18c8

Western blot was performed as previously described (37 (link)). The following primary antibodies, which were purchased from Cell Signaling Technology, were used: Cleaved Caspase-3 (Asp175) (5A1E) (#9664), Cleaved Caspase-8 (Asp391) (18C8) (#9496), p21 (#2947), p27 (#3686), AKT (pan) (40D4) (#2920), Phospho-AKT (Ser473) (D9E) XP (#4060), and Bim (#2933). EGFR (#18986-1-AP), LXR-α (#14351-1-AP), LXR-β (#60345-1-Ig), CHOP (#15204-1-AP), FOXO3A (10849-1-AP), and GAPDH (#60004-1-Ig) were purchased from Proteintech. FLAG was obtained from Sigma-Aldrich (# F1804). All secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).

For direct IB analysis, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with phosphatase inhibitors. The following primary antibodies were used against: cleaved caspase-3 (Asp175) (5A1E) (#9664), cleaved caspase-8 (Asp391) (18C8) (#9496), RB (#9309), p-RB (Ser870/811) (#8516), CDK2 (#2546), p-CDK2 (#2561), Bcl-2 (#15071), Bax (#5023), p21 (#2947), p27 (#3686), cyclin D1 (#55506), cyclin E (#4136), p44/42 MAPK (Erk1/2) (137F5) (#4695), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (#4370), Akt (pan) (40D4) (#2920), phospho-Akt (Ser473) (D9E) XP (#4060), PI3 kinase p110α (C73F8) (#4249), cyclin A2 (#91500), and cyclin B1 ( #4135), all purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to CDK4 (#11026-1-AP), CDK6 (#14052-1-AP), SKP2 (#15010-1-AP), p53 (1C12) (#2524), P16-INK4A (#10883-1-AP), EGFR (#18986-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#60004-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). The anti-FLAG was obtained from Sigma-Aldrich (St. Louis, MO, USA). All secondary antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.

For protein extraction, the cells were harvested after 24 h of prednisolone treatment and sonicated on ice in PRO-PREP™ Protein Extraction Solution (Cat No. 17081, iNtRON BIOTECHNOLOGY, Inc, Korea). A total of 30 µg protein per sample was fractionated by Tris-Glycine-PAG, SDS Precast gel, 8~16% (Cat No. KG75505, Komabiotech, Seoul, Korea), transferred to Immobilon-P membrane, PVDF (Cat No. IPVH00010, Millipore, Corporation Canada), and then blotted with antibodies against GR (ab55189, Abcam, Cambridge, UK), cleaved caspase-8 (Asp391, 18C8, Cell Signaling Technology, Danvers, MA), caspase-9 (Cell Signaling Technology), and β–actin (sc-130656, Santa Cruz Biotechnology, Santa Cruz, CA).