Rabbit anti daxx

The following antibodies were used for immunofluorescence studies: rabbit anti-Daxx (Sigma, D7810), rabbit anti-ATRX H-300 (Santa Cruz, Sc15408), rabbit anti-PML (Bethyl, A301167A), rabbit anti-EZH2 (Cell Signaling, 4905S), rabbit anti-H3K27me3 (Active motif, 39155), mouse anti-ORC2 (MBL, M0553), and mouse anti-αTubulin/Alexa488 (Invitrogen, 322588). Secondary antibodies AlexaFluor488 or AlexaFluor594 were purchased from Invitrogen. The following antibodies were used for Western blotting: mouse anti-ORF50 (provided by Erle Robertson, UPENN), mouse anti-ORF45 (provided by Yan Yuan, UPENN), rat anti-LANA (Advanced Biotechnologies Inc., 13210), rabbit anti-Daxx (Sigma), and anti-actin-HRP (Sigma, A23852). Antibodies used in ChIP assay include: rabbit polyclonal antibodies to histone H3K4me3 (Millipore, 07473), histone H3K27me3 (Active motif,391155), total histone H3 (Bethyl), ORC2 (MBL, M0553), Rad21 (Abcam, ab992), CTCF (Millipore, 07729), or rabbit IgG (Santa Cruz Biotechnology, sc-2027), and rat polyclonal anti-LANA (Advanced Biotechnologies Inc.).

RFP-LANA WT and MT iSLK cells were washed 2 times with cold PBS, and then lysed in cold lysis buffer (20 mM Tris, pH 8.0, 137 mM KCl, 1 mM EDTA, 1.5 mM MgCl₂, 10% Glycerol, and 1% Triton X-100 supplemented with 1 mM DTT and 0.1% mammalian protease inhibitor cocktail mix) for 30 mins on ice. Cell lysates were centrifuged at 13000 rpm for 10 mins, and the supernatants were precleared with Protein G Sepharose beads (GE Healthcare) for 60 mins at 4 °C with rotation. One ml of precleared lysates (~5 x 10⁶ cells) were immunoprecipitated with either rat anti-LANA (Advanced Biotechnologies Inc.) or mouse monoclonal anti-p53 (CalBiochem) or rabbit anti-DAXX (Sigma) overnight at 4 °C with rotation. The immuno-complex was collected with Protein G Sepharose beads with rotating at 4 °C for 3 hrs, and the beads were washed 3 times with BC300 (300 mM KCl, 20 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 10% glycerol, and 10 mM β-mercaptoethanol) followed by once with BC100 at 4 °C. Pulled down proteins were eluted by boiling with 2x Laemmli buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% Bromophenol Blue, and 20% Glycerol), and were subject to SDS-PAGE and Western blot analysis.

The following antibodies were used for immunofluorescence, Co-IP, and Western blotting studies: rat polyclonal anti-HHV8 or anti-LANA [LN53] (Abcam, ab4103), rabbit anti-Coilin (Cell Signaling technology,14168S), rabbit anti-DAXX (Sigma, D7810), rabbit anti-PML (Bethyl, A301167A), mouse anti-ORF50 (provided by Erle Robertson, UPENN), mouse anti-ORF45 (provided by Yan Yuan, UPENN), mouse anti-EBV (BIO-RAD, 4260–0906), anti-RAD21 (Abcam ab992), rabbit polyclonal anti-PARP1 (Enzo ALX-210-302-R100), rabbit EZH2 (Cell Signaling, 4905), rabbit H3K27me3 (Active Motif, 39155), rabbit IgG (Santa Cruz Biotechnology), AlexaFluor594 or AlexaFluor488 (Invitrogen), and mouse anti-Actin-HRP (Sigma, A23852).