Precise protein gel

Equal quantities of protein samples were resolved on 4–20% Precise Protein Gels by SDS-PAGE (4–20% Precise Protein Gels, Thermo Fisher Scientific) and transferred to nitrocellulose membrane (Millipore). Membranes were blocked for 1 hour in 5% nonfat dry milk in tris buffered saline (TBS) with 0.2% Tween 20 and incubated in primary antibodies overnight at 4 °C. Incubations were performed with the following primary antibodies: dystrophin C-terminal (NCL-Dys2, 1:100, Leica Biosystems) and GAPDH (MAB374, 1:50,000, Chemicon). Horseradish peroxidase–conjugated anti-rabbit IgG and anti-mouse IgG (GE Healthcare) secondary antibodies were used at 1:5,000 dilutions in 5% nonfat dry milk and incubated at RT for 3 hours. Immunoblots were developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific).

NeutrAvidin agarose beads (100 µl of 50% slurry, Thermo Scientific) were washed in CCT lysis buffer, which comprised 25 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, 20 mM EDTA, 0.1% v/v Tween-20, and protease inhibitors (Roche). Then, 100 µg of the biotinylated LOX-1 peptide or the control scrambled peptide was added to 100 µl of the resuspended beads, and the CCT lysis buffer was used to bring the final volume to 1 ml. After an overnight incubation at 4°C, the peptide-bound beads were washed once in CCT lysis buffer, and the nonspecific binding sites were blocked with 1 ml of FBS during an overnight incubation at 4°C. The blocked beads were then washed and resuspended in 1 ml of CCT lysis buffer. Purified endogenous CCT (100 µg) from bovine testis was then combined with the peptide-bound beads and incubated overnight at 4°C in the presence or absence of ATP (0.1 mM). After the incubation, the beads were washed 5 times with CCT lysis buffer and collected by centrifugation at 700g for 2 minutes. Collected beads were heated at 95°C in 50 µl of 2× SDS sample buffer for 5 minutes and centrifuged at 13,000g for 1 minute. The eluted proteins (20 µl) were then separated by 4–20% Precise™ protein gels (Thermo Scientific), and western blot analysis was performed to detect CCT1. Purified CCT was run on the same gel as a control.

HUVECs were treated with or without 10 µg/ml of OxLDL (770252-7, Kalen Biomedical, Montgomery Village, MD) for 1 hour, lysed by using the previously described method in section 2.3, and then incubated overnight at 4°C with 1 µg of anti-LOX-1 monoclonal antibody (sc66155, Santa Cruz Biotechnology, Dallas, TX) or isotype-specific (IgG1) control antibody (0102-01, SouthernBiotech, Birmingham, AL). Next, 20 µl Protein-G beads (GE Healthcare, Pittsburgh, PA) prewashed in PIPES buffer was added to the cell lysate and rotated for 2 h at 4°C. The beads were then washed 5 times with PIPES lysis buffer, and the proteins were eluted by adding 2× SDS sample buffer (40 µl) and heating the beads at 95°C for 5 minutes. Eluted proteins were separated by 4–20% Precise™ protein gels (Thermo Scientific), and western blot analysis was performed by using anti-CCT1 (sc53454, Santa Cruz Biotechnology) or anti-CCT4 (sc137094, Santa Cruz Biotechnology) antibodies.