Rabbit anti mllt11

HEK293 cells were transfected with myc or myc-MLLT11 vectors using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, cells were lysed on ice with Tris-HCl lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and protease inhibitor cocktail (Sigma). Lysed cells were collected, centrifuged, and supernatant used for co-IP. Anti-c-Myc Agarose resin (Pierce) was used, as per manufacturer's protocol, to co-immunoprecipitate myc or myc-MLLT11 and their binding partners. Proteins were eluted from the resin with 50 mm NaOH, neutralized with 1 m Tris, pH 9.5, and added to nonreducing sample buffer for Western blot analysis. For whole-brain lysates, E18.5 cortical protein samples were separated on 8% SDS-PAGE gels for 1 h at 120 V and transferred overnight at 20 V on to PVDF membranes (Bio-Rad). Blots were probed with mouse anti-acetylated tubulin (1:20,000, Sigma), and rabbit anti-Mllt11 (1:2000, Abcam). Secondary antibodies were goat anti-rabbit HRP (1:5000, Invitrogen) and goat anti-mouse HRP (1:5000, Invitrogen). Blots were developed with Clarity Western ECL Substrate (Bio-Rad) and imaged on a ChemiDoc Touch Gel Imaging System (Bio-Rad). Band densitometry was done using Image Lab Software (Bio-Rad).

Cortices were microdissected from E18.5 embryos, digested in trypsin (Pierce), manually triturated and plated on 35-mm well onto poly-D-lysine coated coverslips at a density of 150,000 cells for the neurite outgrowth assay or 500,000 for cellular localization experiments. Cells were plated in medium containing DMEM with 10% FBS and 1% penicillin/streptomycin and 4 h after plating, media was completely removed and replaced with Neurobasal media containing B-27+ (Gibco/Invitrogen), 1% penicillin/streptomycin, and L-glutamine. Cells were cultured for 24 h or one week in a 37°C incubator containing 5% CO₂, then fixed for 10 min in 4% paraformaldehyde. Immunocytochemistry was conducted using the following antibodies: mouse anti-Tau (1:200; Abcam), rabbit anti-MAP2 (1:1000; Abcam), rabbit anti-Mllt11 (1:300, Abcam), rabbit anti-Tuj1 (Tubb3; 1:1000, Biolegend), and mouse anti-acetylated α-tubulin (1:1000, Sigma).