N dodecyl β d maltoside

For measurement of enzyme activities, frozen skeletal muscle samples were pulverized in liquid nitrogen (BioPulverizer, BioSpec Products, Inc., Bartlesville, OK), homogenized in sucrose homogenization buffer (20 mM Tris, 40 mM KCl, 2 mM EGTA, and 250 mM sucrose, pH 7.4) with 0.05% detergent (n-Dodecyl β-D-maltoside; Sigma D4641), sonicated (F60 Sonic Dismembrator, Fisher Scientific, Waltham, MA), and centrifuged at 11,000 g for 3 min. The supernatant was stored at −80 °C until further analysis of citrate synthase (CS) and cytochrome c oxidase (CCO) activities.
Enzymatic activities were measured as previously described^{17 ,33 (link)} using a microplate reader (Synergy HT, BioTek Instruments, 237 Winooski, VT, USA). An 80-fold dilution of muscle homogenate was analyzed for CS and CCO activities. Briefly, CS activity was assessed at 412 nm by measuring the linear rate of reaction of free CoA-SH with DTNB; CCO activity was determined by measuring the linear rate of oxidation of fully reduced cytochrome c at 550 nm.
Enzyme activities in muscle homogenates were normalized to protein content, determined using the Bradford Protein Assay Kit (Thermo Scientific, Rockford, IL). Using CS activity as a proxy for total mitochondrial content in the sample^{34 (link)}, CCO activity was further normalized to mitochondrial content.

Blue native-polyacrylamide gel electrophoresis (BN-PAGE) of integral thylakoid proteins was performed as previously described [36 ]. Five grams of fresh leaf tissues were homogenized in liquid nitrogen and thylakoid membranes were extracted using an extraction buffer (pH 7.8) containing 20 mM Tricine-NaOH, 70 mM sucrose, and 5 mM MgCl₂ and were filtered through miracloth/cheesecloth before centrifugation at 4500× g for 10 min. The thylakoid pellet was resuspended in the same buffer (pH 7.8) and centrifuged again. The resulting pellet containing thylakoid membranes was washed and extracted with each proper buffer. An equal volume of resuspension buffer containing 2% (w/v) n-dodecyl-β-d maltoside (Sigma-Aldrich, St. Louis, MO, USA) was added under continuous mixing and the solubilization of membrane-protein complexes was allowed to occur for 3 min on ice. Insoluble material was removed by centrifugation at 18,000× g for 15 min. The supernatant was mixed with 0.1 volume of 5% w/v Serva blue G, 100 mM Bis Tris-HCl (pH 7.0), 30% w/v sucrose, and 500 mM €-amino-n-caproic acid and loaded onto a 0.75-mm-thick 5%–12.5% w/v acrylamide gradient gel (180 × 160 mm). Electrophoresis was performed at 4 °C by increasing the voltage from 100 to 200 V overnight.

Blue Native one-dimensional electrophoresis was performed for identification of whole respiratory complexes in isolated mitochondrial fractions of left ventricles of NMX and HPX offspring hearts. Isolated mitochondrial membrane fractions were isolated as previously described. The pellet was solubilized with 0.1 mM N-Dodecyl β-D-maltoside (Sigma-Aldrich, St. Louis, MO) similar to that for activity assays. To separate the respiratory chain complexes, 30 μg of mitochondrial protein was loaded onto a high-resolution Clear Native PAGE of 3-12% Bis-Tris gradient gel (Thermo Fisher Scientific). The gel was stained with Coomassie dye and destained according to standard methods. The bands of individual complexes were visualized by the ChemiDoc Touch Imaging System (Bio-Rad).

n-Dodecyl β-D-maltoside (DDM), dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, acetonitrile, and formic acid were obtained from Sigma-Aldrich (St. Louis, MO). Promega trypsin gold was purchased from Promega Corporation (Madison, WI). Synthetic heavy peptides labeled with ¹³C/¹⁵N on the C-terminal arginine or lysine were purchased from New England Peptide (Gardner, MA).

As previously described [46 (link), 51 (link), 52 (link)], mitochondrial outer membrane integrity was evaluated by the measurement of cytochrome C oxidase (COX) activity in mitochondrial fractions in the presence and absence of detergent n-dodecyl β-D-maltoside (Sigma-Aldrich, Saint Louis, MO, Catalog Number CYTOCOX1). According to manufacture’s protocol, 20 μg freshly isolated mitochondrial fraction was used for each reaction. COX activity was measured by its oxidation of substrate ferrocytochrome C. Mitochondrial outer membrane damage was assessed from the ratio between the activity with and without n-dodecyl β-D-maltoside present (1 μM) under each experimental condition [53 (link)]. All measurements were performed in triplicate.

Retinal tissue of α-crystallin B and PBS injected eyes was carefully separated from the parts used for IHC staining and transferred into individual 2 mL sample tubes in liquid nitrogen. The tissues were firstly disintegrated with a precooled mortar and subsequently lysed using 0.5% N-Dodecyl β-d-maltoside (Sigma Aldrich) in PBS for one hour at 4 °C. Further breakdown of the retinal cells was facilitated using an ultrasonic bath for 30 min on ice and multiple impulses of an ultrasonic wand. The protein concentration for each sample was determined with a BCA Pierce Protein Assay kit (Thermo Fisher, Waltham, MA, USA), and 10 µg of the total protein mixture was transferred into 1× LDS sample buffer (NuPAGE, Thermo Fisher) containing 0.1 M DTT. Each sample was subsequently boiled at 70 °C for ten minutes and separated on a 4–12% NuPAGE Novex Bis-Tris precast gel (Life Technologies, Waltham, MA, USA) for ten minutes at 180 V in 1× MOPS buffer. The individual gel lanes were further cut into one piece per lane and were destained with 50% ethanol and 25 mM ammonium bicarbonate (ABC). After dehydration with 100% acetonitrile (ACN), the gel pieces were digested using trypsin at 37 °C overnight. The tryptic peptides were extracted twice with 3% TFA and 30% CAN, concentrated with an Eppendorf concentrator and passed through a C₁₈ Stage Tip [56 (link)].