Sap18

For immunofluorescent staining, isolated cerebral tissues were mounted in OCT and used for preparing cryosections. Cryosections (10 μm) were fixed and permeated with acetone or 4% paraformaldehyde with 0.5% Triton X-100, and then incubated with antibodies against Pnn (P3A, 1:1000, kind gift from Dr. Pin Ouyang), SAP18 (1:100, Santa Cruz), a neuron nuclear antigen (NeuN) (1:500, Abcam), and GFAP (1:1000, Cell Signaling Technology) at 4 °C overnight. Sections were then incubated with Alex488 or Alex594-conjugated goat anti- mouse or rabbit invitrogen (IgG) for 1 h at room temperature. After counterstaining with DAPI, sections were examined with a fluorescent microscope.

HSPCs were chemically cross-linked with a 1% formaldehyde solution for 10 min at room temperature with gentle agitation and quenched with 0.125 M glycine. The fixed cells were resuspended, lysed, and sonicated to solubilize and shear the crosslinked DNA. The samples were ultra-sonicated for 8 min with 30 s ultra-sonication at 30 s intervals. The resulting fragmented chromatin extract was precleared with protein A/G beads (ThermoFisher, Beijing, China) and then incubated separately overnight with SAP18 (Santa Cruz Biotechnology) or mSin3A (Santa Cruz Biotechnology) antibodies followed by washing, elution, and reverse cross-linking. DNA was purified using PCR purification kits (QIAGEN, Hilden, Germany) and amplified by quantitative PCR using the specific primer sets as listed below for individual genes. ChIP-qPCR calculations were performed as described previously^{52 (link)}. In brief, the protein-specific Ab ChIP-ed DNA signal intensity value was divided by the intensity value of the IgG-ChIP-ed signal, representing the fold enrichment of the protein on the specific region of genomic DNA. The primer sequences used are listed in Supplementary Table 4.

Rabbit polyclonal anti-eIF4A3, anti-Y14, and anti-MLN51 are gifts from C. Tomasetto. Rabbit polyclonal anti-Acinus is from Bethyl (A300-999A) and goat polyclonal Pinin is from Santa Cruz. Rabbit polyclonal SAP18 is from Santa Cruz Biotechnology (sc-25377). Rabbit polyclonal GAPDH is from Cell Signalling.
Plasmids for recombinant protein expression were previously described^{3 (link),24 (link)}.
The original p3xFLAG-CMV-eIF4A3 was a gift from M. Moore. p3XFLAG-CMV-PininWT (siRNA-resistant) was created by site-directed mutagenesis (Invitrogen) for regions targeted by siRNA Pinin. The mutant Pinin with RBS deletion (aa 237–246) was generated from the p3xFLAG-CMV-PininWT. The HA-Acinus was generated by cloning the Acinus S’ cDNA into pcDNA 3.1 vector.