Control mouse igg

ChIP-PCR was performed as previously described (26 (link)). MDA-MB-231 cells were treated for 24 hours with vehicle or 500 nM JQ1, and chromatin was immunoprecipitated with an anti-BRD4 specific antibody (Bethyl Laboratories, A301-985A50) or a control mouse IgG (Sigma, I5281). The following promoter-specific primers were used: FOXM1, forward 5ʹ-GTAAGATGGAGGCGGTGTTG-3ʹ and reverse 5ʹ-GGGTGGCCTACCTTCTTAGG-3ʹ; E2F2, forward 5ʹ-GACAATAGCAGGCACCCAGTA-3ʹ and reverse 5ʹ-AGCACTGGATTGCGAGTCTG-3ʹ; E2F8, forward 5ʹ-TAGGAAGCACCCACCTGTTC-3ʹ and reverse 5ʹ-GGGAGAAATCCAGGCATCTA-3ʹ; LIN9, forward 5ʹ-GGAACTGCAGGCTGTTTGTT-3ʹ and reverse 5ʹ-GGGTTTCGGGAACTGTGAGT-3ʹ; MYBL2, forward 5ʹ-GTCTTCAAGTCCCAGCCAGT-3ʹ and reverse 5ʹ-CCGGAATGTTAAGGAGCAAA-3ʹ.

At the collection time cells were treated with trypsin to remove exogenous X-DING and sample was divided into two portions used in parallel for intra- and extracellular staining. For intracellular X-DING detection, cells were fixed with 1% paraformaldehyde (PFA) for 20 minutes at room temperature (RT); while cells subjected to extracellular staining were processed without the permeabilization step (see below). Subsequently, cells were pelleted and resuspended in PBS supplemented with 1% saponin (Sigma-Aldrich), 1% FBS and 0.1% NaN₃, then placed in V-shaped 96-well plates, pelleted by centrifugation and washed twice with PBS / 1% saponin. Cells were stained with mouse monoclonal antibody to X-DING at 1:250 dilution or control mouse IgG (Sigma-Aldrich) at 1:500 dilution; following 30 minutes incubation at RT and washing in PBS, cells were stained for 30 minutes with the secondary anti-mouse Alexa Fluor 488 antibody (Life Technologies) at 1:150 dilution. After the subsequent washing, cells were resuspended in PBS / 1% saponin for X-DING analysis. Cells for control IgG staining were harvested at 30 minute time-point. MFI was used to calculate statistical significance.

Cells were washed once in PBS and once in a sample buffer (50mM Tris pH7.4, 150mM NaCl, 0, 1% NaN₃ and 2% FBS). Cells were stained for 30 min in RT with mouse phycoerythrin (PE)-conjugated monoclonal antibody to CD4 at 1:250 dilution, mouse monoclonal antibody to X-DING at 1:250 dilution or control mouse IgG (Sigma-Aldrich) at 1:500 dilution. Samples stained with anti-X-DING required additional 30 min staining with secondary anti-mouse Alexa Fluor 488 antibody as described above. Subsequently samples were washed in sample buffer then were resuspended in PBS / 1% saponin for X-DING analysis.
Concentration of the intra- and extracellular X-DING protein and extracellular CD4 was acquired by C6 flow cytometer (AccuriCytometers, Inc) and analyzed by FlowJo software (Tree Star Inc.). The gating was set manually to show percentages of X-DING positive cells excluding the interfering background and debris events.

Samples were generated before and after immunoprecipitation in the presence or absence of RNase I as described^{5 (link)} using 5–10 μg anti-FMRP, anti-p-UPF1 S1116, control rabbit immunoglobulin (IgG) (Sigma–Aldrich) or control mouse IgG (Sigma–Aldrich) per 0.5 ml lysate at 1–2 mg protein per ml. When determining immunoprecipitation efficiencies, 5% of immunoprecipitated samples was used in western blotting. When assaying for co-immunoprecipitated proteins, 20% of immunoprecipitated samples was used in western blotting.