Ea03752box

Samples were homogenized with RIPA buffer supplemented with protease inhibitor cocktail. Lysate supernatants were quantified using a Pierce BCA protein assay kit (Thermo Fisher Scientific, 23225) and adjusted to an identical concentration using H₂O. Equal amounts of sample were mixed with NuPAGE LDS sample buffer (Invitrogen, NP0007) and 10% β-mercaptoethanol and then boiled at 70°C for 10 minutes. Ten micrograms total protein per lane was loaded into 3%–8% Tris-acetate gels (Invitrogen, EA03752BOX) and electrophoresed for 1 hour at 200 V. Protein was transferred onto a PVDF membrane under wet conditions at 350 mA for 3.5 hours. The membrane was blocked in 5% nonfat milk in TBST buffer and then incubated with primary antibody to label the specific protein. After washing 3 times with TBST, the membrane was incubated with HRP-conjugated secondary antibody specific to the IgG of the species of primary antibody against dystrophin (MilliporeSigma, D8168) or vinculin (Cell Signaling Technology, 13901S). The target proteins were visualized using chemiluminescent substrates (Invitrogen, WP20005).

Muscle samples were homogenized with RIPA buffer supplemented with protease inhibitor cocktail. Lysate supernatants were quantified with Pierce BCA protein assay kit (Thermo Fisher Scientific, 23225) and adjusted to an identical concentration using H₂O. Samples were mixed with in NuPAGE LDS sample buffer (Invitrogen, NP0007) and 10% β-mercaptoethanol followed by boiled at 70°C for 10 min. 20 μg total protein per lane was loaded into 3 to 8% tris-acetate gel (Invitrogen, EA03752BOX) and electrophoresed for 1 hours at 200 V. Protein was transferred on a PVDF membrane under the wet condition at 350 mA for 3.5 hours. The membrane was blocked in 5% non-fat milk in TBST buffer and then incubated with primary antibody labeling specific protein. After washing three times with TBST, the membrane was further incubated with HRP conjugated secondary antibody (1:1000 dilution, Beyotime, A0216) specific to the IgG of the species of primary antibody against dystrophin (1:1000 dilution, Sigma, D8168) and vinculin (1:1000 dilution, CST, 13901 S). The target proteins were visualized with Chemiluminescent substrates (Invitrogen, WP20005).

Lysates were loaded onto NuPAGE 4–12% 15-well Bis-Tris gels (Invitrogen; NP0323BOX), NuPAGE^TM 10% Bis-Tris, 1.0 mm, 10-well protein gels (ThermoFisher Scientific; NP0301BOX), or NuPAGE 3–8% Tris acetate gels (Invitrogen; EA03752BOX) and run at 200 V for 55 min or 150 V for 60 min, respectively. Gels were wet transferred using 20% methanol onto either NuPAGE nitrocellulose 0.2-μm pore sheets (Invitrogen; LC2000) or Odyssey® nitrocellulose membranes, 0.22 μm, 7 × 8.5 cm (P/N 926-31090). Membranes were blocked for 1 h at room temperature in Odyssey Blocking Buffer (TBS) (LI-COR 927-50000) or the stated blocking solution. Primary antibodies were diluted with their respective blocking buffers (see figure legends) and incubated overnight at 4 °C. Washes were performed with TBS 0.1% Tween 20 (TBS-T) before addition of secondary antibody for 1 h at room temperature. Washes were performed with 1× TBST before imaging. For demonstration of equal protein loading following multicolor imaging, membranes were incubated with HRP-conjugated GAPDH primary antibody (ab9385) for 1 h at room temperature in Odyssey Blocking Buffer (TBS). Membranes were washed with TBS-T before staining was developed using Optiblot ECL Detect kit (ab133406) and imaged on a GBOX XT-16 chemiluminescent imager with a 20-min exposure.

SDS-PAGE was performed under both reducing and non-reducing conditions. The rHA samples were mixed with 2× Laemmli buffer (Bio-Rad, 161–0737), and approximately 4-5 μgs of rHA was loaded per lane. For reducing conditions, a final concentration of 100 mM DTT was added to the Laemmli-rHA solution using a 500 mM DTT stock (Pierce, product# 20291, lot# ND170603) and incubated in a 100°C heat block for 3–5 min prior to loading on to the gel. Non-reduced and reduced samples were separated using either 3-8% Tris-Acetate gels (Life Technologies, EA03752BOX) and Tris-Acetate SDS Running Buffer (Invitrogen, LA0041) or 4-12% NuPAGE Bis-Tris Gels (Cat# NP0323, Life Technologies Corporation, Carlsbad, CA) and 1× MES Running Buffer (50 mM MES, 50 mM Tris, 0.1% sodium dodecyl sulfate, 1 mM EDTA pH 7.3) at 150 volts for 1 hour. Protein bands were visualized by Coomassie staining (Bio-Rad, 161–0787). Gels were stained for 1 hour followed by a water wash until protein bands developed. Alternatively, purity and trypsin resistance gels were stained for 1 hour in 0.1% Brilliant Blue R, 7.7 M reagent alcohol, 1.75 M glacial acetic acid, and de-stained in 10% acetic acid. Gels were scanned using Gel Logic 212 Pro Imaging System, and densitometry was performed using the Carestream Molecular Imaging software (Carestream Health, Incorporated, New Haven, CT).

Isolated tissues were homogenized with RIPA buffer supplemented with protease inhibitor cocktail. Adjust lysate supernatants to an identical concentration using H2O, after quantifying it with Pierce BCA protein assay kit (Thermo Fisher Scientific, 23 225).
For DMD^Q1392X mice, equal amounts of samples were mixed with in NuPAGE LDS sample buffer (Invitrogen, NP0007) and 10% β‐mercaptoethanol and then were boiled at 70°C for 10 min. 10 µg total protein per lane was loaded into 3 to 8% tris‐acetate gel (Invitrogen, EA03752BOX) and electrophoresed for 1 h at 200 V. Protein was transferred to a PVDF membrane under wet condition at 350 mA for 3.5 h. The PVDF membrane was incubated with primary antibody labeling specific protein after blocking in 5% non‐fat milk in TBST buffer. The membrane was further incubated with HRP conjugated secondary antibody specific to the IgG of the species of primary antibody against dystrophin (Sigma, D8168) and vinculin (CST, 13901S), after washing three times with TBST. The target proteins were visualized with Chemiluminescent substrates (Invitrogen, WP20005). The gray values were quantified by using ImageJ.