Mp imaging system

The EMSA was conducted using 50 nM of 5’ 6-Fluorescein (5’ 6-FAM) pT labelled DNA (hybridized or not with the complementary non-fluorescent DNA strain to form ssDNA and dsDNA) from IDT. LMNG was used as a detergent for TraE, while Triton X-100 was used for TraD. Proteins were diluted in 10 mM Tris pH 9.5, 150 mM NaCl, 10% glycerol, 5x detergent CMC and incubated with DNA for 30 min at 4 °C. Samples were loaded on a 0.5% agarose gel and run in the running buffer: 0.5X Tris-Borate-EDTA (TBE) pH 9.5, 150 mM NaCl, 5% glycerol, 5x detergent CMC at 90 V, 4 °C for 2 h. Agarose gels were prepared by solubilizing agarose with the running buffer, and gel revelation was acquired using a Fluorescein filter from the ChemiDoc MP imaging system. For BAR-072 inhibition experiments, all solutions and gels were supplemented with 10% DMSO.

PBMCs were isolated from prepared buffycoats in a standard Ficoll gradient procedure to develop a target engagement assay for MKK4. Isolated PBMCs were preincubated with 0.3, 1 and 3 μM HRX215 in RPMI (1% P/S, 10% FCS) at 37° C for 2 hours. Afterwards activation of the MAPK signaling pathway was induced by the addition of 1 μg/ml LPS for 1 h. Harvested cells were lysed with NP-40 Buffer (50 μM TrisHCl, pH7.4, 150 mM NaCl, 0.5% NP-40 including protease and phosphatase inhibitors) and total protein concentrations were determined according to the DC Protein Assay Instruction Manual.
The following immunoblotting was performed as described above with an anti-p-MKK4 (S257) antibody (CST4514). A ChemiDoc MP Imaging System was used to visualize p-MKK4 levels. Intensity of protein bands were quantified via ImageJ and analyzed in GraphPad Prism.

Mouse muscle lysates were prepared using 4 M urea lysis buffer (pH 6.8); 150 μL of lysis buffer was added to ten sections of 20-μm-thick tissue. Tissue was lysed using a metal bead (2 min at 30 Hz, TissueLyser II; Qiagen). The solution was incubated at room temperature for 30 min prior to a second lysis step (1 min at 30 Hz, TissueLyser II). The solution was incubated again at room temperature for 30 min. The solution was then centrifuged at 14,000 × g for 20 min and supernatant extracted for analysis. Protein was quantified using a Bio-Rad DC assay kit (catalog #5000112). A calibration curve was made by spiking in WT dystrophin from C57Bl/6 mice into dystrophin-null lysate from a Dup2Del18-41 mouse. All samples were mixed with 4× Laemmli buffer prior to incubation at 95°C for 5 min. Thirty micrograms of total protein was loaded on a precast 3%–8% tri-acetate gel and ran for 1 h at 80 V followed by 2 h at 120 V. Gels were transferred overnight at 4°C, constant 55 mA onto a 0.45-μm polyvinylidene fluoride membrane. Membranes were cut at 150 kDa marker for probing. The top halves of membranes were probed using a polyclonal rabbit antibody specific to the C terminus of dystrophin (ab15277, 1:200 or ab154168, 1:1,000; Abcam). Membranes were then washed four times for 5 min in 0.1% Tween 20 containing PBS (PBST). Secondary antibody goat anti-rabbit HRP (1:5,000) for 1 h at room temperature followed by five 5-min washes with PBST and one 5-min wash with PBS. Membranes were incubated with 2 mL of ECL reagent (Thermo Scientific, #34580) prior to visualization on a Chemidoc MP Imaging System. Dystrophin signals were quantified using Image Lab software (version 6.0.0 build 25). Individual mouse samples were quantified using a linear regression curve fitted to a calibration curve on each gel. The full-length (427 kDa) and IRES-driven (413 kDa) dystrophin isoforms could not be differentiated on the western blots.