Anti bcl xs l

Cells were lysed with the M-PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) containing a protease inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4–12% gradient or 12% SDS-PAGE gels, followed by transfer to polyvinylidene fluoride membranes. After blocking membranes, blots were incubated with the indicated primary antibodies: anti-Bcl-2 (sc-492; Santa cruz biotechnology [SCB], Dallas, Texas, USA, anti-Bcl-X_S/L (#633901; BioLegend, San Diego, CA, USA ), anti-Mcl-1 (sc-819; SCB ), anti-caspase-3 (9668; Cell Signaling Technology [CST], Danvers, MA, USA ), anti-caspase-8 (M032-3; Medical and Biological Laboratories, Nagoya, Japan), anti-caspase-9 (9508; CST), anti-β-actin (BioLegend ) or anti-α-tubulin (SCB). After washing, room temperature incubation of membranes for 30 min with either goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Invitrogen) were used to detect the primary antibodies. Protein bands were visualized using an ImageQuant LAS-4000 system (FujiFilm, Tokyo, Japan).

Cells were lysed with a mammalian protein extraction reagent (M-PER; Thermo Scientific, Rockford, IL, USA) containing a protease-inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4–12% gradient or 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked and the blots incubated with the following primary antibodies: anti-Bcl-2 (sc-492; Santa cruz biotechnology (SCB), Dallas, Texas, USA, anti-Bcl-X_S/L (#633901; BioLegend, San Diego, CA, USA), anti-Mcl-1 (sc-819; SCB), anti-FLIP_S/L (sc-5276; SCB), anti-DR5 (#8074; Cell signaling technology (CST), Danvers, MA, USA), anti-CHOP (#2895; CST), anti-caspase-3 (#9668; CST), anti-caspase-8 (M032–3; Medical and Biological Laboratories, Nagoya, Japan), anti-caspase-9 (#9508; CST), anti-Bid (#2002; CST), anti-β-actin (BioLegend), or anti-α-tubulin (SCB). After washing, room temperature incubation of membranes for 30 min with either goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Invitrogen) was used to detect the primary antibodies. Protein bands were visualized using CDP-star chemiluminescence and imaged using an ImageQuant LAS-4000 system (FujiFilm, Tokyo, Japan).