Ab92446

The following primary antibodies were used: α‐BAF (PU38143, a generous gift from T. Haraguchi), α‐LEMD2 (HPA017340, Sigma), α‐SATB2 (ab92446, Abcam), α‐V5 epitope tag (R960‐25, Thermo Fisher Scientific), α‐Lamin B2 (33‐2100, Thermo Fisher Scientific), α‐Fos (226004, Synaptic Systems), α‐Lamin B1 (ab16048, Abcam), α‐VPS4 (E‐8) (sc‐133122, Santa Cruz), α‐phospho‐MSK1 (Thr581) (#9595, New England Biolabs), α‐ERK2 (C‐14) (sc‐154, Santa Cruz), α‐GAPDH (MAB374, Millipore), and α‐beta‐III Tubulin (NB100‐1612, Novus Biologicals).
Secondary antibodies were as follows: goat anti‐mouse Alexa‐488 (A11001, ab150117), goat anti‐mouse Alexa‐555 (A21422), donkey anti‐mouse Alexa‐488 (A21202), goat anti‐rabbit Alexa‐555 (A21428), goat anti‐rabbit Alexa‐488 (ab150081), donkey anti‐rabbit Alexa‐555 (ab150062), and donkey anti‐goat Alexa‐555 (A21432).

Neonatal cortical tissue was homogenized using a dounce homogenizer in IP lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP‐40, 1 mM EDTA, 5% glycerol; Pierce). DIV13 cortical cultures were lysed on the culture dish in IP lysis buffer (Pierce). The lysates were incubated for 10 min on ice while shaking, followed by a brief centrifugation at 13,000 g. The supernatant was used in immunoprecipitation reactions using the Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 50 µl of protein G Dynabeads was coated with 5 µg of anti‐SATB2 antibody (ab92446, Abcam), and beads were mixed with 500 µg of cortical lysate and incubated overnight at 4°C. On the next day, the beads–antibody–protein complexes were washed three times with washing buffer, re‐suspended in 2 × Roti‐Load sample buffer (Roth) for elution, and incubated at 95°C for 5 min. The eluates were separated by SDS–PAGE and used for further immunoblotting analysis.

The following primary antibodies were used: anti-SATB2 (ab92446, Abcam); anti-SATB1 (ab92307, Abcam); anti-HDAC1 (10E2) (5356P, Cell signaling); anti-HNRNPL (ab156682, Abcam); anti-HNRNPL-like (4783S, Cell signaling); anti-HNRNPC1/C2 (M022726, BOSTER); anti-ZNF638 (orb215138, Biorbyt); anti-DHX9 (PA5-19542, Thermo Scientific); anti-CUX1 (M-222) (sc-13024, Santa Cruz); anti-Lamin A/C (N-18) (sc-6215, Santa Cruz); anti-ERK2 (C-14) (sc-154, Santa Cruz), anti-BANF1 (PU38143, a generous gift from T. Haraguchi [48 (link)]; anti-pan LAP2 (Lap2 2–12), a generous gift from R. Foisner [49 (link)], anti-CHMP3 (HPA015673, Atlas Antibodies).

Cell lysates were prepared in 1x SDS Sample Buffer (62.5 mM Tris-HCl pH 6.8, 2%SDS, 42 mM DTT, 10% glycerol and 0.01% bromophenol blue; Cell Signaling Technology), sonicated to shear DNA and reduce viscosity and then heat denatured. Proteins were separated on 4%–20% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% milk and incubated with primary antibodies for 1 h at room temperature. Then the membranes were incubated with following primary antibodies at appropriate dilution in blocking buffer: ant-SATB1 (ab109122, Abcam 1: 10,000), anti-SATB2 (ab92446, sc-81376, 1:2000), anti-CDX2 (Abcam, 1:5,000), anti-OCT4 (sc-5279, Santa Cruz Biotechnology, 1:2,000), anti-GATA4 (sc-25310, Santa Cruz Biotechnology, 1:2,000), anti-FLAG (#14793, Cell Signaling Technology, 1:5,000) and ELF5 (sc-9645, Santa Cruz Biotechnology, 1:2,000). Anti-TUBA (MABT522, Millipore Sigma, 1:20,000), anti-ACTB (A5441, Millipore Sigma, 1:30,000) or anti-Histone H3 (ab1791, Abcam, 1:20,000) antibodies were used detect the expression of housekeeping genes as loading controls. Membranes were washed, blocked, and incubated with peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat secondary antibodies (Santa Cruz Biotechnology) at a dilution of 1:5,000–2,0000, and immunoreactive signals were visualized using Luminata Crescendo Western HRP substrate (Millipore Sigma).

The following antibodies were used: rabbit anti-bromodeoxyuridine (1:1000; catalog #12304, Megabase Research Products); rat anti-Ctip2 (1:500; catalog #ab18465, Abcam); rabbit anti-Cux1 (1:300; catalog #sc-13 024, Santa Cruz Biotechnology); rabbit anti-Cux1 (1:1000; catalog #11733–1-Apr, Proteintech); rabbit anti-doublecortin (DCX; 1:1000; catalog #ab18723, Abcam); goat anti-GFP (1:500; catalog #600–101-215 M, Rockland); rabbit anti-Ki67 (1:500; catalog #AB9260, EMD); goat anti-Mcm2 (minichromosomal maintenance protein 2; 1:250; catalog #sc-9839, Santa Cruz Biotechnology); rabbit anti-Pax6 (1:500; catalog #GTX113241, GeneTex); mouse anti-phospho vimentin (1:500; catalog #D076-3, MBL); mouse anti-RFP (1:200; catalog #LS-C29691, LSBio); rabbit anti-Satb2 (1:2000; catalog #ab92446, Abcam); rabbit anti-Sox9 (1:1000; catalog #AB184547, Abcam); rabbit anti-Tle4 (1:500; catalog #ab140485, Abcam); and rabbit anti-Tbr2 (Eomes; 1:500; catalog #AB23345, Abcam). Fluorescent secondary antibodies were used at 1:1000 (Jackson ImmunoResearch).