Las imagequant system

The procedure was carried out as previously described [45 (link)]. Briefly, tissue or cultured CPCs were lysed in RIPA buffer (1% NP-40, 1% SDS, 0.5% sodium deoxycholate diluted in Phosphate Buffered Saline, PBS) supplemented with protease inhibitor (cOmplete Protease Inhibitor Cocktail, Roche) and sonicated for 10 cycles of 10 sec sonication and 10 sec pause with the Bioruptor Next Gen Sys (Diagenode, Seraing, Belgium). After centrifugation (10 min, 13000 rpm) the supernatant was collected. Protein concentrations were determined with Bradford reagent (Bio-Rad). 15 μg of protein extract were loaded with 5x Laemmli buffer on Mini Protean TGX gels (Bio-Rad) and run at 100V for 1.5 h. Proteins were transferred to PVDF membranes (Trans-blot Turbo Transfer Pack) using the Trans-blot Turbo Transfer System (both from Bio-Rad) following the manufacturer's instructions. Membranes were blocked with 5% BSA in TBS-T (blocking buffer) for 1 h and incubated overnight with primary antibodies (diluted in blocking buffer). Membranes were washed, incubated with secondary antibodies for 1 h and detected using ECL or Femto substrates (Thermo Scientific) and LAS ImageQuant System (GE Healthcare, Little Chalfont, UK).
The following antibodies were used: anti-FOXG1 (1:1000 dilution; #18529) and GAPDH (1:3000 dilution; #8245), from Abcam. For densitometric analyses, ImageJ software was used [52 ].

mESC or NPC48h were lysed in RIPA buffer (1% NP-40, 1% SDS, 0.5% sodium deoxycholate diluted in Phosphate Buffered Saline, PBS). Cells were centrifuged (10 min, 13,000 rpm) and the supernatant collected. Protein concentrations were determined with Bradford reagent (Bio-Rad). Fifteen micrograms of protein extract were loaded with 5x Laemmli buffer on Mini Protean TGX gels (Bio-Rad) and run at 100 V for 1.5 h. Proteins were transferred to PVDF membranes (Trans-blot Turbo Transfer Pack) using the Trans-blot Turbo Transfer System (both from Bio-Rad) following manufacturer’s instructions. Membranes were blocked with 5% BSA in TBS-T (blocking buffer) for 1 h and incubated overnight with primary antibodies (diluted in blocking buffer). Membranes were washed, incubated with secondary antibodies for 1 h and detected using ECL or Femto substrates (Thermo Scientific) and LAS ImageQuant System (GE Healthcare, Little Chalfont, UK). Antibodies details are listed in Supplementary Table 3. For densitometric analyses, ImageJ software was used⁵⁵ . The code used for the analysis of densitometric data is available at https://github.com/FrancescoFerrari88/code_DOT1L_paper/blob/master/figure_3/NOTEBOOKS/3a-3b_EPZ_vs_DMSO_H3K79me2_GLOBAL_BULCK_%26_LOCUS-SPECIFIC_CHANGES.ipynb.