Creb1 antibody

Total proteins were isolated from cells with lysis buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP40; 2.5 mM sodium pyrophosphate; 0.02% sodium azide; 1 mM EGTA, 1 mM EDTA; 1 mM β-glycerophosphate; 1 mM Na₃VO₄; 1 mM PMSF; 1 μg/ml leupeptin). The lysates were centrifuged at 12 000r.p.m. for 30 min at 4 °C. The protein concentration was determined by Bradford dye method. Equal amounts (20–50 μg) of cell extract were subjected to electrophoresis in 8–12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and transferred to PVDF or nitrocellulose membranes (Millipore, Darmstadt, Germany) for antibody blotting. The membranes were blocked and then incubated with CREB1 antibody (all from Cell Signaling Technologies, Boston, MA, USA), Aromatase, ERα, ERβ and NR5A2 antibody (all from Abcam, Cambridge, UK), β-actin antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at 4 °C. Subsequently, the membranes were incubated with an HRP-conjugated anti-mouse or -rabbit secondary antibody (Protein Tech Group, Chicago, IL, USA) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence reagent (ECL) kit (GE Healthcare, Munich, Germany), according to the manufacturer's instructions. Blots were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).^{48 (link)}

Co‐IP assay was performed in accordance with our previous study(Huang et al., 2019 (link)). In brief, after the supernatants of brain samples were collected, the lysates were incubated with MeCP2 antibody (Santa Cruz Biotechnology, Texas, USA) and protein A/G agarose gel overnight, followed by SDS‐PAGE resolution and Western blot assay. CREB1 antibody (Cell Signaling Technology, Inc., Danvers, USA) was used to measure the interaction of the two proteins.

WT and AKIP1-depleted HepG2 cells were transfected with the plasmids pCAGGS-NP (1.25 μg), pCAGGS-VP35 (1.25 μg), pCAGGS-VP30 (0.75 μg), pCAGGS-L (10 μg), pCAGGS-T7 (2.5 μg), and p4cis-vRNA-RLuc (2.5 μg). After 48 h, the cells were lysed and assayed with the ChIP-IT High Sensitivity kit (53040, Active Motif, USA) as previously described. The CREB1 antibody (Cell Signaling Technology, 9197, 1:25) was used to precipitate potential bound viral RNA. Precipitated RNA was reverse transcribed and quantified using real-time PCR. Primer pairs from EBOV 3′ leader (3Le, 1-469) and 5′ trailer (5Tr, 18283-18959) as described previously were employed^{15 (link)}. The PCR primers are shown in Supplementary Table 1.