Image studio light software

Mouse brains (brain development) for each development time period were used to prepare western blot samples as per [65 (link)]. Nuclear extracts of HEK293 cells expressing the 3 × Flag versions of E1 and E2 were used to prepare serial dilutions and calculate MeCP2-E1/MeCP2-E2 ratios. Frozen frontal cortices (circadian) and cell pellets (CHX) were homogenized on Laemmli buffer 3% B-Mercaptoethanol. In all experiments, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [66 (link)] and transferred onto nitrocellulose membranes (GE Healthcare), and blocking by incubating in 5% skimmed milk in PBS with 0.1% Tween 20. Membranes were incubated with specific antibodies, either overnight at 4 °C or for 1 h at room temperature. Antibodies and dilutions used were as follows: MeCP2 1:5000, β-actin 1:20,000, Histone H4 1:20,000 Histone H3 1:20,000 (Abcam), Flag 1:5000 (Sigma-Aldrich), EGFP 1:1000 (Thermo Fisher), in house rabbit polyclonal antibodies MeCP2 E1 1:1000 and E2 1:500. Fluorescent secondary antibodies were (Li-Cor) 1:10,000. Densitometries were performed with Li-Cor Image Studio Light software.

Cells were lysed in 1× Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM dithiothreitol [DTT], and 0.02% bromophenol blue), and cell lysates were homogenized with QIAshredder columns (Qiagen). Samples were boiled at 95°C for 5 min before they were loaded on Bolt 4 to 12% Bis-Tris plus gels (Thermo Fisher Scientific) to perform protein separation by gel electrophoresis. After 20 to 35 min at 200 V, proteins were transferred to nitrocellulose membranes (GE Healthcare Life Sciences) for 70 min at 10 V. Next, membranes were blocked in 5% milk in Tris-buffered saline (20 mM Tris-HCl [pH 7.6] and 150 mM NaCl) with 0.1% Tween 20 (Sigma-Aldrich) (TBS-T) for 1 h at room temperature (RT) before membranes were incubated with appropriate dilutions of primary antibodies in TBS-T either for 2 h at RT or overnight at 4°C. After incubation with conjugated secondary antibodies in TBS-T, signals were developed using an LAS-4000 mini (Fujifilm Life Sciences) or an Odyssey Fc imaging system (LI-COR Biosciences). Signal intensities were quantified using the Image Studio Light software (LI-COR Biosciences).

For Western blot analysis, lysates were boiled in Laemmli sample buffer with ß-mercaptoethanol (4% (v/v)). Following SDS-PAGE proteins were transferred to nitrocellulose by electroblotting (Mini Trans-Blot^® Cell, BioRad). Membranes were blocked by incubation in TBS-T (TBS with 0.1% (w/v) Tween 20) containing 5% skimmed milk for 1 h at room temperature and incubated with primary antibody in TBS-T + 5% skimmed milk overnight at 4 °C. After washing with TBS-T blots were incubated with respective secondary antibody (IRDye-Infrared Technology Licor) in TBS-T for 1 h at room temperature. Protein signals were visualized using ODYSSEY^® 9120 Scanner and Image Studio Light software (Licor).

Cells were lysed in 1× Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM dithiothreitol [DTT], and 0.02% bromophenol blue), and cell lysates were homogenized with QIAshredder columns (Qiagen). Samples were boiled at 95°C for 5 min before they were loaded on Bolt 4 to 12% Bis-Tris plus gels (Thermo Fisher Scientific) to perform protein separation by gel electrophoresis. After 20 to 35 min at 200 V, proteins were transferred to nitrocellulose membranes (GE Healthcare Life Sciences) for 70 min at 10 V. Next, membranes were blocked in 5% milk in Tris-buffered saline (20 mM Tris-HCl [pH 7.6] and 150 mM NaCl) with 0.1% Tween 20 (Sigma-Aldrich) (TBS-T) for 1 h at room temperature (RT) before membranes were incubated with appropriate dilutions of primary antibodies in TBS-T either for 2 h at RT or overnight at 4°C. After incubation with conjugated secondary antibodies in TBS-T, signals were developed using an LAS-4000 mini (Fujifilm Life Sciences) or an Odyssey Fc imaging system (LI-COR Biosciences). Signal intensities were quantified using the Image Studio Light software (LI-COR Biosciences).