Sc 514008

Proteins were extracted with RIPA lysis buffer (Thermo Fisher Scientific, Inc., Waltham, MA). Anti-p113 polyclonal antibody was prepared by immunizing rabbits with synthesized peptide corresponding to C-terminus of p113 (EQQLSAKNSTLKGRRD; ABclonal Biotechnology Co., Ltd., Wuhan, China). Western blot analysis was performed as previously described [14 (link)–16 (link)], with antibodies specific for CUX1 (ab230844), transcription factor 3 (TCF3, ab69999), ALDH3A1 (ab186726), NDUFA1 (ab249923), NDUFAF5 (ab240971), β-actin (ab179467, Abcam Inc., Cambridge, MA), CUX1 (sc-514,008), histone H3 (sc-517,576), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-47724), glutathione S-transferase (GST, sc-33614, Santa Cruz Biotechnology, Santa Cruz, CA), Flag-tag (14793S), ZRF1 (12844S), BRD4 (13440S), hemagglutinin (HA)-tag (3724S), or His-tag (12698S, Cell Signaling Technology Inc., Danvers, MA).

Tissue or cellular protein was extracted with 1× cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described (Zhang et al, 2012; Zhao et al, 2016; Li et al, 2018b), with antibodies (1:500 dilution) specific for CUX1 (sc‐514008, Santa Cruz Biotechnology, Santa Cruz, CA), ENO1 (ab155102), GPI (ab66340), PGK1 (ab38007), EWSR1 (ab93837), ELAVL1 (ab136542), SYNCRIP (ab184946), MAZ (ab85725), MUC4 (ab60720), S100A9 (ab92507), KLF10 (ab73537), TXNIP (ab188865), AMPKβ1 (ab32112), β‐actin (ab8227), Flag (ab18230), Myc (ab9106, Abcam Inc., Cambridge, MA), GST (sc‐33614), or histone H3 (sc‐10809, Santa Cruz Biotechnology).

Mice were anesthetized with a 4:1 cocktail of ketamine and xylazine (Bayer) and perfused transcardially with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 m PBS. Brains were removed, postfixed for 6 h in 4% paraformaldehyde, and incubated overnight in 0.1 m PBS containing 30% sucrose. Cryosections (30 μm thick) were mounted on SuperFrost Plus glass slides for immunofluorescence analysis. Tissue sections were washed (10 min) in PBS; incubated in blocking solution containing 0.5% Triton X-100, 4% horse serum, and PBS (1 h, room temperature); and incubated overnight at 4°C in blocking solution containing the first primary antibody. Tissue was then washed in PBS (10 min), followed by incubation in secondary antibody for 1 h at room temperature. The primary antibodies used were as follows: anti-Tbr1 (1:500, chicken polyclonal; catalog #AB2261, Millipore); anti-CaMKIIa (1:500; mouse; catalog #SA-162, Biomol Research Laboratories); anti-Sim1 (1:1000; rabbit; catalog #ab4144, Millipore; RRID:AB_2187608); anti-Cux1 (1:100; mouse; catalog #sc-514008, Santa Cruz Biotechnology). The secondary antibodies used were as follows: Alexa Fluor 488 donkey anti-mouse (RRID:AB_141607); Alexa Fluor 647 donkey anti-chicken (RRID:AB_11194678); and Alexa Fluor 647 donkey anti-rabbit (RRID:AB_2536183; all diluted 1:1000).