Ecl western blot detection system

Following Hcy treatment, nuclear extracts of HRECs and ARPE-19 were prepared using a nuclear extraction kit purchased from Abcam (ab113474) (Abcam Inc., Cambridge, MA, USA) to detect nuclear levels of NF-κB. Both nuclear extracts and cytoplasmic extracts were subjected to gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The protein was blotted to nitrocellulose membranes, which were further blocked using 5% milk solution and then incubated with the NF-κB antibody (1:300, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Membranes were then re-probed with histone deacetylase (HDAC) (Abcam Inc., Cambridge, MA, USA) as the loading control for nuclear extracts, and actin (Abcam, ab5694, Cambridge, MA, USA) as the loading control for cytoplasmic extracts. Blots were then incubated with an appropriate peroxidase-conjugated secondary antibody. An enhanced chemiluminescence (ECL) western blot detection system (Thermo Scientific) was used for visualization of protein bands and ImageJ software was used to determine the optical density of the bands. Data are presented as ratio of nuclear to cytoplasmic NF-κB. Samples were representative to at least three mice for each experiment.

EVs isolated from conditioned medium of C2C12 cells were labeled labeled with the membrane dye DiR (Xenolight, Perkin Elmer) following manufacturers recommendations. DiR is a near-infrared lipophilic dye that binds the lipid bilayer surrounding EVs [20 (link)]. Its emission spectra is only visible using infrared CCD imaging. We injected 100 µl of DiR dye alone or DiR-labeled EVs at 1.0 mg/ml and then imaged (Ex =710, Em=790) 24 hrs later using an Ami X spectral imaging instrument. Bone marrow cells were flushed from long bones of young adult mice and adherent and non-adherent cells cultured and treated (50µg/ml EVs) with normal C2C12 EVs and miR-34a overexpressed EVs for 18 hrs. The cell lysate was prepared for western blot analysis as per our published method [57 (link)]. In brief, protein was extracted from cell culture lysate, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated with a polyclonal antibody against Sirt1 (Millipore Anti-Sirt1 Cat # 07-131), and beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 ºC, followed by incubation with appropriate secondary antibody. Proteins were visualized with an ECL Western blot detection system (Thermo Scientific, Waltham, MA).

Extracellular vesicles were lysed in RIPA buffer containing proteinase inhibitor cocktail (Sigma) followed by quantification of total protein concentration using a Bradford assay (Bio-Rad Laboratories) according to the manufacturer’s protocol. Equivalent amounts of total lysate were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with an antibody against CD81, CD63 and TSG101 (Santa Cruz, CA) overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody. Proteins were visualized with an ECL Western blot detection system (Thermo Scientific, Waltham, MA).

Protein was extracted from quadriceps muscle and cell culture lysate, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated with a polyclonal antibody against glutathione peroxidase (GPx1), superoxide dismutase (SOD2) (Santa Cruz Biotechnology, Santa Cruz, CA), NOX2, 3-NT, eNOS (Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by incubation with an appropriate secondary antibody. Proteins were visualized with an ECL western blot detection system (Thermo Scientific, Waltham, MA). For detection of eNOS dimers, we ran a low-temperature SDS-PAGE (LT-PAGE) gel using reported procedures [20 (link)] with slight modification. The protein lysates were prepared using 1× Laemmli buffer without 2-mercaptoethanol. The samples were then subjected to SDS-PAGE with 7.5% gel and run at a low temperature by keeping the buffer tank surrounded by ice. The gels were transferred, and the blots were probed as described above.

Cells were washed and lysed with radio-immunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor to extract the total protein. An equal concentration of protein was separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After being blocked in tris-buffered saline with tween^® 20 (TBST) containing 5% non-fat dry milk for at least 1 h at room temperature, the membranes were incubated with specific primary antibodies (1:1000 for rabbit anti-Nox2; 1:1000 for rabbit anti-Nox4; 1:500 for mouse anti-p22; and 1:1000 for mouse β-actin) for at least 16 h at 4 ℃. Next, the membranes were washed three times and incubated with a peroxidase-conjugated secondary antibody (1:10,000 for anti-rabbit or anti-mouse IgG), for 1 h. Finally, the bands were developed using an enhanced chemiluminescence (ECL) western blot detection system (Thermo Fisher Scientific), and the relative protein expression was quantified using Image Lab 6.0 software (Bio-Rad, Hercules, CA, USA) by the gray value.

Protein was extracted from retinal tissues and concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL). 20 μg of protein were subjected to SDS–PAGE, transferred to nitrocellulose membranes, and then incubated with primary antibodies overnight at 4 °C. Secondary detection was done using horseradish peroxidase– conjugated secondary antibodies (Promega, Madison, WI). After washing, the proteins were visualized using enhanced chemiluminescence (ECL) western blot detection system (Thermo Fisher Scientific). β- actin served as the loading control. Western blot analysis was repeated twice with comparable results.