Rabbit anti sox2

Seeded NSCs were washed with PBS and fixed with ice-cold 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.5% Triton X-100 in TBS for 5 min and blocked with 2% FBS in TBS for 30 min at room temperature. The cells were incubated with primary antibodies: rabbit anti-sox2 (1:200, cell signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-β-tubulin III (1:200, Millipore, MA, USA), rabbit anti-GFAP (1:200, Stem Cell Technologies, BC, CA), rabbit anti-phosphorylated IRF3 (Ser396) (1:200, cell signaling, CA, USA), or mouse anti-ZIKV E protein (1:500, GeneTex, CA, USA) at 4 °C overnight. The cells were then washed three times with TBS and incubated with Dylight 594-conjugated donkey anti-mouse secondary antibody or Dylight 488-conjugated goat anti-rabbit secondary antibody (1:1000, Jackson ImmunoResearch Laboratories, PA, USA) for 1 h at room temperature. The cells were then washed three times with TBS, and the cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, MO, USA). The images were collected with a fluorescence microscope (Olympus BX51, Olympus, Tokyo, JP).

Cell samples were lysed in a buffer containing 150 mM NaCl, 10 mM HEPES, 2 mM MgCl₂, 10 mM KCl, 0,5 mM EDTA and 0.5% Triton X-100. Protease inhibitors were added freshly. The Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher) and equal amounts of protein were separated by electrophoresis on SDS/PAGE and blotted onto cellulose membranes (BioTrace NT, Pall). Membranes were blocked with 3% BSA in TBST and then incubated with the primary antibody over night at 4 °C (i.e. sheep anti-Trim71 (R&D Systems) rabbit anti-Ago2 (Cell Signaling) rabbit anti-TuJ1 (Cell Signaling), rabbit anti-Sox2 (Cell Signaling), mouse anti-Tubulin (Sigma).
After washing with TBST, followed by incubation with HRP-conjugated secondary antibodies (Jackson ImmunoResearch), bands were detected by chemiluminescence (Thermo Scientific).

The cultured cells were washed with cold PBS and then treated with cell lysis buffer (2× loading buffer, 2 μg·mL⁻¹ Aprotinin, 1 mm PMSF, 2 mm β‐mercaptoethanol) at 100 °C for 10 min. Then the mixture was centrifuged under 4 °C at 14000g for 10 min. Approximately 10 μL of protein was loaded in each lane of 10% SDS/PAGE gel and separated, and the protein then transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight, followed by the secondary antibody. The antibodies used were rabbit anti‐CD133 (Cell Signaling Technology, Danvers, MA, USA; #64326), rabbit anti‐OCT4 (Cell Signaling Technology; #2750), rabbit anti‐SOX2 (Cell Signaling Technology; #3579), rabbit anti‐NANOG (Cell Signaling Technology; #4903), mouse anti‐KRAS (Santa Cruz Biotechnology, Dallas, TX, USA; sc30), mouse anti‐β‐Tubulin (Cell Signaling Technology; #6181), rabbit anti‐TET3 (Cell Signaling Technology; #85016), rabbit anti‐Lin28B (Signalway Antibody LLC, College Park, MD, USA；#21626), rabbit anti‐PKCβ (Cell Signaling Technology; #46809), rabbit anti‐PKCδ (Cell Signaling Technology; #2058), rabbit anti‐PKCζ (Cell Signaling Technology; #9372), rabbit anti‐PKCμ (Cell Signaling Technology; #2056) and mouse anti‐Flag (Sigma; CAT F1804).