T5192

Mouse monoclonal antibodies used were anti-GFP ([1:100] Sigma-Aldrich, G6539), acetylated tubulin ([1:1,000] Sigma-Aldrich, T7451) and anti-histone H2B ([1:50] Abcam, ab52484) antibodies. Rabbit polyclonal antibodies used were anti-γ-tubulin ([1:1,000] Sigma-Aldrich, T5192), anti-lamin A/C ([1:20] Santa Cruz Biotechnology, H110), anti-cAMP protein kinase catalytic subunit ([1:1,000] Abcam, ab26322) and anti-CBX/HP1 beta antibodies ([1:100] Abcam, ab10478). TRITC or Alexa Fluor 647 conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc. or Life Technologies, respectively) were used for indirect immunofluorescence detection. BG-conjugated dyes, including SNAP-Surface Alexa Fluor 647 and 546 (NEB), were used for staining SNAP-tag expressed in cells.
The cells were fixed with 4% formaldehyde (Electron Microscopy Sciences) for 15 min, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS), stained for primary antibodies and Alexa-Fluor-conjugated secondary antibodies, or BG-conjugated fluorophores for SNAP-tag at 4 °C overnight.

Cell lysate preparation and protein detection was performed as described [11 (link)]. Briefly, protein lysates were prepared from harvested cells, separated by electrophoresis using a Tris-acetate gel, transferred to PVDF membrane, and the membrane was probed for protein using the antibodies indicated in each figure. Antibodies used: ATRX (Sigma, HPA001906 or GeneTex, GTX629703), DAXX (Sigma, HPA008736), γ-tubulin (Sigma, T5192) and Ku70 (Santa Cruz, sc-9033).