Anti daxx

Cells were grown on No. 1.5 glass coverslips for 24 h with complete medium followed by any indicated treatments. Fixatives, permeabilization solutions, and antibodies were prepared in phosphate buffered saline (1 X PBS). Coverslips were fixed for 10 min in 4% formaldehyde. Coverslips were washed in PBS three times for 5 min each. Cells on the coverslips were then permeabilized in a 0.1% Triton X-100 for 10 min. Cells were then incubated with primary antibodies for 1 h at room temperature and then washed three times in PBS for 5 min each and incubated with secondary antibodies for 1 h at room temperature. Antibody dilutions were: anti CK2 1:100 (Thermo Scientific), anti V5 1:100 (Millipore), anti ZC3H8 1:100 (Assay Biotech), anti COILIN 1:100 (monoclonal, Abnova) or 1:100 (Thermo, polyclonal), anti PML 1:2000 (Acris), anti DAXX 1:25 (Cell Signaling), anti SMN1 1:100 (Cell Signaling), and anti NARG2/ICE2 1:100 (Bioss). Cells were washed three more times in PBS for 5 min each then mounted on slides with Vectashield +DAPI and sealed with nail polish. Images were captured and processed using a Leica SP8 confocal microscope system with a Leica 63X oil immersion objective.

GSK-3β inhibitor VIII, N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea, was purchased from Calbiochem (San Diego, CA, USA). Cell Counting Kit-8 (CCK-8) was from Dojindo (Tokyo, Japan). Mitochondria isolation kit for cultured cells and RIPA buffer were obtained from Thermo Scientific (Rockford, IL, USA). Annexin V and propidium iodide (PI) for flow cytometry were purchased from BD Pharmingen (San Diego, CA, USA). For the Western blot, the following specific antibodies were used: anti-tau (Invitrogen, Carlsbad, CA, USA), anti-phospho-tau (Ser396) (Invitrogen, Carlsbad, CA, USA), anti-Fas (Santa Cruz Biotech, Delaware, CA, USA), anti-Fas ligand (Santa Cruz Biotech, Delaware, CA, USA), anti-cleaved caspase-8 (Novus Biologicals, Littleton, CO, USA), anti-p38α (Santa Cruz Biotech, Delaware, CA, USA), anti-Daxx (Cell signaling, Beverly, MA, USA), anti-cleaved caspase-3 (Cell Signaling, Beverly, MA, USA), and anti-cytochrome C (Cell Signaling, Beverly, MA, USA).