Cd95 fas

Total cell lysates were prepared by sonicating and boiling cell pellets in 1× Laemmli reducing sample buffer. To prepare detergent-soluble lysates, cells were lysed in cold isotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 0.1% Triton X-100), 150 mM NaCl, with proteases inhibitor cocktail and 1 mM PMSF for 15 minutes on ice and centrifuged for 10 minutes at 20,000 g. The clarified supernatant was used as the detergent soluble cell fraction. Equal volumes of cellular lysate or equal protein amounts were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membranes. Proteins were detected by immunoblotting.
Antibodies used in this study were purchased from the following sources: anti-actin (Sigma-Aldrich); anti-ubiquitin clone P4D1, goat, anti-rabbit/mouse/rat IgG-conjugated horseradish peroxidase, USP7, USP5, USP9×, USP24, UCHL-5, USP14 and OUTB1 (Bethyl Laboratories; anti–poly (ADP-ribose) polymerase (PARP), Cleaved PARP (Asp214), Caspase8, Caspase3, BID, BAX (Cell Signaling Technology); anti-HA (clone 3F10; Roche Applied Science), anti-NOXA Santa cruz and CD95/Fas (Clone EPR5700; Epitomics)).

In this study, sinularin was obtained from National Museum of Marine Biology & Aquarium (Pintung, Taiwan) and the chemical structure showed in Figure 1B. The anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA), apoptosis antibody sample kit, pro-apoptosis bcl-2 family antibody sample kit, pro-survival Bcl-2 family antibody sample kit, phospho-pI3 kinase p85 (Tyr458)/P55 (Tyr799) antibody, phosopho-GSK3β (Ser9) (D3A4) rabbit antibody, PI3 kinase p110α antibody were obtained from Cell Signaling (Danvers, MA, USA), anti-cytochrome C, CD95/Fas, AIF, phospho-Akt1, Akt1, mTOR/FRAP and phosopho-mTOR (Phospho-Ser2481) antibody were obtained from Epitomics (Bellerica, MA, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained for Millipore (Bellerica, MA, USA).

Total cell lysates were prepared by sonicating and boiling cell pellets in 1X Laemmli reducing sample buffer. Detergent-soluble cell lysates were prepared by lysing cells in cold isotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, along with protease inhibitor cocktail and 1 mM PMSF for 15 minutes on ice and centrifuged for 10 minutes at 20,000 g. The clarified supernatant was used as the detergent soluble cell fraction. Lysates were electrophoresed (SDS-PAGE gels) and transferred to nitrocellulose membranes (Whatmann). Proteins were detected by immunoblotting.
Antibodies used in this study were purchased from the following sources: anti-actin and FLAG (Sigma-Aldrich); anti-ubiquitin clone P4D1, goat, anti-rabbit/mouse/rat IgG-conjugated horseradish peroxidase, p21, ERK and Mcl-1 (Santa Cruz Biotechnology); USP7, USP5, BRAF and p73 (Bethyl Laboratories); anti–poly(ADP-ribose) polymerase (PARP), pERK, Caspase8, Caspase3, Bid, Bax, Bak (Cell Signaling Technology); anti-HA (clone 3F10; Roche Applied Science); CD95/Fas (Clone EPR5700; Epitomics); anti-NOXA, p53 (DO-1; CalBiochem); anti-DR4, DR5 (ProSci incorporated) and FAS monoclonal antibody (CH11; Millipore).