Anti sox2

Tissues from two offspring/litter and three to four litters/treatment group (n = 6–8 pups/treatment group) were used for all in vivo immunohistochemistry experiments. Tissue was coronally sectioned using a Leica cryostat at 30 μm thickness and serially collected. Animal numbers were randomized for blinded counting. The primary antibodies used for staining of select cortical markers for E18.5 samples were anti-GFP 1:1000 (Aves Lab, Tigard, OR, CAT#GFP-1020), anti-Ki67 (Abcam, Cambridge, MA, CAT#ab15580), anti-Sox2 1:1000 (Abcam, Cambridge, MA, CAT#ab97959), anti-Tbr2 1:1000 (Millipore, Billercia, MA CAT#AB15894), anti-RELN 1:1000 (Invitrogen, CAT#PA5–47537), anti-CUX1 1:100 (Atlas Antibodies, St. Louis, MO, CAT#HPA003277), anti-Ctip2 1:300 (Abcam, Cambridge, MA, CAT#ab187668), and anti-SATB2 1:1000 (Abcam, Cambridge, MA, CAT#ab92446). The primary antibody used for staining of neuronal populations of PND70 samples was anti-TH (Abcam, Cambridge, MA, CAT#ab76442). Primary antibody incubation occurred at 4° C overnight, following which sections were incubated with secondary antibodies (goat anti-chicken 488, CAT#A11039, goat anti-rabbit 568, CAT#A11011 and goat anti-mouse 568, CAT#AB175473). Sections were then washed and incubated with DAPI nuclear stain (Thermo-Fisher, CAT#D1306).

hESCs were grown on Matrigel-coated coverslips. Cells were fixed with the 4% paraformaldehyde solution containing 0.5% Triton X-100. Fixed cells were incubated with the blocking solution (3% BSA in PBS containing 0.1% Tween 20) for 1 h. Cells were then treated with primary antibodies diluted in the blocking solution and incubated overnight at 4C. Primary antibodies used for staining included anti-ZO-1 (Invitrogen), anti-SOX2 (Abcam), anti-NANOG (Thermo Fisher Scientific), anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX1 (Cell Signaling), anti-KI67 (Abcam), anti-γ-tubulin (Abcam), anti-acetylated tubulin (Sigma-Aldrich), anti-ARL13B (Proteintech).
The slides were then washed with PBS containing 0.1% Tween 20, stained with secondary antibodies and DAPI and mounted with the vectashield antifade mounting medium (Vector Laboratories). The slides were viewed with a DeltaVision microscope (GE healthcare). Sum stack of image slices was used for intensity quantification. ImageJ was used to determine the signal intensities of nuclei. Normalized signal intensities were calculated by subtracting background signals from nuclear signals.

Cell pellets were incubated with 1X SDS sample buffer and then lysed by sonication. Lysates were cleared by centrifugation and analyzed by SDS-PAGE followed by Western blotting. The following primary antibodies were used: anti-p53 DO-1 (Santa Cruz Biotechnology) that recognizes epitopes in the region containing residues 11–25, anti-p53 pAB1801 (Santa Cruz Biotechnology) that recognizes epitopes in the region containing residues 32–79, anti-p53 C-terminal antibody (Sigma-Aldrich) that recognizes epitopes in the region containing residues 334–383, anti-GAPDH (Cell Signaling Technology), anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX2 (Abcam), anti-NANOG (Santa Cruz Biotechnology), anti-Tubulin (Millipore Sigma) and anti-BBS9 (Millipore Sigma). Secondary antibodies used were anti-rabbit or anti-mouse IgG (H + L) (Dylight 680 or 800 conjugates) (Cell Signaling Technology). The membranes were scanned with the Odyssey infrared imaging system (LI-COR Biosciences).