Ab10480

Cos-7 cells were seeded onto 10-cm² dishes the day before transfection. The following plasmids psg5-V5-PRMT5, pcdna3.1-HA-HP1γ, and pcdna3.1-GR were transfected into Cos-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 48 h after transfection, cells were treated (or not) with Dex for 24 h, and cell extracts were prepared in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.25% deoxycholate) supplemented with protease inhibitor tablets and phosphatase inhibitors (1 mM NaF, 1 mM Na₃VO₄, and 1 mM β-glycerophosphate). Protein extracts were incubated with HP1γ primary antibody (ab10480; Abcam), PRMT5 (ab109451; Abcam) or GR/DGH2L (#12041; Cell signaling) over night at 4°C under agitation. Protein A Agarose (Millipore) beads were then added, and the mixture was incubated 2 h at 4°C. The immunoprecipitates were separated on SDS–PAGE. Immunoblotting was conducted with primary antibodies against GR G-5 (sc-393232; Santa Cruz), GR/D6H2L (#12041; Cell signaling), HP1γ (ab10480; Abcam), and PRMT5 (07-405; Millipore). Secondary antibodies were used for chemiluminescence detection using the ECL detection reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions. For immunoprecipitation experiments, 3% of the input of each sample was analyzed by immunoblot.

The primary antibodies against the following proteins were used: GFP at 1:3000 (Abcam, ab290, ab69314), NIPBL I at 1:1000 (Enervald et al., 2013 (link)), NIPBL II at 1:500 (Santa Cruz, sc-374625) (Zuin et al., 2014 (link)), α-tubulin at 1:1000 (Sigma, T9026), γH2AX at 1:1000 (Millipore, 05-636), MAU2 at 1:200 (Visnes et al., 2014 (link)), HP1γ at 1:1000 (Millipore, 05-690 and Abcam, ab10480), RAD18 at 1:2500 (Abcam, AB57447), cyclin B1 at 1:400 (Santa Cruz Biotechnology, SC-245), RNF8 at 1:200 (Santa Cruz Biotechnology, SC-271462), RNF168 at 1:1000 (Millipore, ABE367), KAP1 at 1:1000 (Nordic Biosite, A300-274A), KAP1 phosphorylated at S824 at 1:1000 (Nordic Biosite, A300-767A), β-actin at 1:1000 (Abcam, ab8224), Chk1 at 1:1000 (Cell Signaling Technology, #2360), Chk1 phosphorylated at S345 at 1:1000 (Cell Signaling Technology, #2348), DNA-PKcs at 1:1000 (Thermo Fisher, MA5-13404), DNA-PKcs phosphorylated at S2056 at 1:1000 (Abcam, ab18192), poly(ADP-ribose) at 1:1000 (Enzo Life Sciences, 10H). All antibodies were previously validated by us or the respective manufacturer.