Igg rabbit

The InlP polyclonal antibody was obtained after injection of an immunogenic peptide (amino acids 365 to 379, LDVSYNHNYATGGVC) in rabbits and subsequent affinity purification. The InlC polyclonal antibody is described in^{27 (link)}. The other primary antibodies are polyclonal antibodies against RBM5 (Sigma, HPA017335 for IF and Bethyl, A302-228A for IP), PML (Abcam, sc-966), anticoilin (Proteintech, 10967-1-AP), anti-nucleolin (Santa cruz, sc-13057) and monoclonal antibodies against tubulin (hybridoma E7), SC35 (AbCam, ab11826), FLAG-M2 (Sigma-Aldrich, F1804), Myc (9E10, Santa Cruz Biotechnology, sc-40), HA (6E2, Cell Signaling technology #2367), V5 (R960-2, Invitrogen). The immunoprecipitation control antibodies are IgG mouse (Santa-Cruz, sc-2025) and IgG rabbit (Santa-Cruz sc-2027). The secondary antibodies are coupled to Alexa-488 (Life technologies) or Cy3 or Cy5 (Jackson ImmunoResearch). DAPI and Hoechst are from Roche Applied Sciences and Thermo Fisher Scientific, respectively. Lipofectamine LTX Max is from Invitrogen.

ChIP assays were performed as described [33 (link),34 (link)]. Briefly, 2.5 million cells were crosslinked with 37% w/v formaldehyde, quenched with 2.5M glycine, washed 3 times with ice cold PBS, and sonicated in a Bioruptor (Diagenode). The following primary antibodies were utilized for incubation of crosslinked chromatin overnight: HDAC1 (Abcam #31263, 3μg), HDAC2 (Abcam #12169, 3μg) pan-H4ac (Upstate #06–866, 5μg), H3K9Ac (Abcam #12179, 5μg), H3K9me3 (Abcam #8898, 5μg), SIN3A (Abcam #3479, 5μg) and as a negative control IgG Rabbit (Cell Signaling Technologies #2729S, 3 or 5μg), IgG Rabbit (Santa Cruz #2027, 3 or 5μg), or IgG Mouse (Santa Cruz #2025, 3 or 5μg,). Protein A/G magnetic beads (Pierce #88802) were added at 50 μL per ChIP sample. ChIP-qPCR primers were designed using Primer3 web tool and are listed in S5 Table.

Nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, 78833), and lysates were pre-cleared with A/G agarose beads (Millipore, LSKMAGA02) for 1 h at 4 °C. LSD1 antibody (Cell Signaling 2139) at 1:50 dilution and IgG Rabbit (Santa Cruz, 2027) at 1:500 dilution were added to each sample and incubated on a rotator over night at 4 °C. The pulldown of LSD1 antibody and IgG control was achieved by adding A/G agarose beads to the samples and incubating them on a rotator for 3 h at 4 °C. Samples were then used for western blot analysis using the following antibodies: LSD1 (Cell Signaling, 2139) and VDR (Santa Cruz Sc-13133 (Clone D-6)).