Rabbit anti pml

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.).

To assess the effect of HHV-6A infection on the ND10 complex, JJHan cells were infected with HHV-6A-GFP. Cells were fixed with 4% paraformaldehyde (PFA) at 24 h post infection (hpi), permeabilized with 0.1% Triton-X 100 and blocked with 10% BSA. Cells were then stained with rabbit anti-PML (Bethyl Laboratories, Montgomery, TX, USA) and mouse anti-gp82 antibodies (clone 2-D6; HHV-6 foundation repository) at 1:1000 dilutions and further stained using goat anti-rabbit Alexa 568 and goat anti-mouse Alexa 647 antibodies, respectively. Images were acquired on an Andor iXon888 EMCCD using a Nikon-based spinning-disk confocal microscope at 100× magnification (Visitron Systems GmbH, Puchheim, Germany). Images were processed by ImageJ, Adobe Photoshop and Illustrator software (San Jose, CA, USA). One hundred cells were imaged and the number of PML foci per nucleus counted in a blinded manner. The HHV-6A infected cells were further grouped with respect to the stage of infection: (i) immediate early infected cells that only express GFP and (ii) GFP/gp82 double positive indicative of late replication. In addition, immunofluorescence was also used to confirm the knockdown of PML in JJHan cells after lentiviral transduction as described above.

2 × 10⁵ JJHan-KD, 293T-KD, and corresponding control cells were harvested and lysed in a radioimmunoprecipitation assay (RIPA) buffer as described previously [34 (link)]. Proteins were separated on 12% SDS polyacrylamide gels, blotted onto nitrocellulose membranes, blocked and stained with the rabbit anti-PML (Bethyl Laboratories) followed by a HRP-conjugated goat anti-rabbit antibody and the Amersham™ ECL™ Prime western blotting detection kit (GE Healthcare, Chicago, IL, USA). Subsequent experiments were conducted on JJHan clone 6 or 293T-KD clone 2 cell lines.