Peroxidase conjugated secondary antibody

Identical amounts of membrane protein or cell lysate (15-20 µg) were separated on 10% SDS-PAGE gels, followed by transfer to nitrocellulose membranes. Loading was systematically verified using Red Ponceau staining. Immunoblotting was performed as previously described using the different antibodies as indicated [8] (link), [10] (link). For cell lysates, β-tubulin was used as loading control. Protein bands were revealed by chemiluminescence (GE Healthcare, Orsay, France) using a peroxidase-conjugated secondary antibody and a chemiluminescence kit (GE Healthcare), followed by imaging on a Bio-Rad Fusion FX5 (Vilber Lourmat, France). Densitometric analysis was performed using ImageJ software.

Cells samples were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH7.5) with protease inhibitors (Roche, Basel, Switzerland) and 1 mM Na₃VO₄. Protein concentration of cell lysate was estimated by BCA (Thermo Fisher, Bedford, MA, USA). Cell lysates were separated on SDS-PAGE Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes (Life Technologies). The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C. The membrane was washed in TBST and further incubated with peroxidase-conjugated secondary antibody (anti-mouse 1:10,000, anti-rabbit 1:1000, GE Healthcare). The peroxidase activity was detected using either the Amersham ECL Western blotting detection kit (GE Healthcare) or the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermofisher Scientific, Waltham, MA, USA).

After the transfection of siRNA and control, proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer, and the amount of the protein quantified according to a standard assay protocol (DC protein assay, Bio‐Rad, USA). About 50 μg of each sample prepared with SDS‐loading buffer and proteins were separated on NUPAGETM 4%–12% Bis–Tris protein gels (Invitrogen). Each sample from each group (n = 4) were run in duplicates. Then, proteins were transferred to PVDF membranes using Trans‐Blot Turbo transfer system (Bio‐Rad). Membranes were blocked with 5% non‐fat milk in PBS and incubated with the primary antibodies (Table 2) over night. The next day, after washing several times with TBS‐T, membranes were incubated with the appropriate peroxidase‐conjugated secondary antibody (GE Healthcare) for 1 h. Finally, membranes were immersed with the Pierce ECL Western Blotting Substrate (Thermo Scientific), and luminescence was imaged by Fuji Film Las‐1000 and images analyzed using ImageJ.

C2C12 cells were homogenized in Radio-Immunoprecipitation Assay (RIPA) buffer (150 mM NaCl, 1.0% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, and 50 mM Tris-HCl pH8.0) containing a protease inhibitor cocktail (Millipore-Sigma) and phosphatase inhibitors (Millipore-Sigma). 20 μg of cell lysate was resolved using a TGX gel (Bio-Rad, Hercules, CA, USA), transferred to a polyvinylidenedifluoride membrane, and blotted with the appropriate primary antibody. Membranes were incubated with peroxidase-conjugated secondary antibody (GE Healthcare Bioscience, Marlborough, MA, USA). Protein bands were visualized using an enhanced chemiluminescence reagent (Bio-Rad), digitized using a Lumino-image analyzer (LAS-4000 iniEPUV, Fuji Film, Tokyo, Japan or FUSION SOLO. 7S. EDGE., Vilber, France) and quantitated using the program Multi Gauge (Fuji Film) or Evolution Capt (Vilber). Anti-GAPDH (#60004, Proteintech, Rosemont, IL), anti-phospho-Akt (Ser 473) (#4060, Cell Signaling Technology, Danvers, MA, USA), anti-pan-Akt (#4691, Cell Signaling Technology), anti-phospho-mTOR (Ser 2448) (#5536, Cell Signaling), and anti-mTOR (#2983, Cell Signaling Technology) were used as primary antibodies.

Frozen tissues of 10 abdominal aortic intimae including four early lesions and six advanced lesions (S1 Table) were solubilized with lysis buffer (1% Triton X in phosphate buffer saline and 50 mg/mL aprotinin). Then, the quantity of the whole protein was uniformed in each solubilized tissue, using a TaKaRa BCA Protein Assay Kit (bicinchoninate method, a modified Lowry method) (Takara Bio Inc., Shiga, Japan). Proteins were electrophoresed under reducing conditions on 10%–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd., Osaka, Japan). Separated proteins were transferred to a nitrocellulose membrane. After blocking the nonspecific background with 4% skim milk, the membrane was incubated with a primary antibody against C1q (clone 9A7; Abcam, Tokyo, Japan) or β-actin (clone AC-74; Sigma-Aldrich Corp., St. Louis, MO, USA), followed by incubation with a peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK). Immunoreactive bands were visualized using ECL reagents (GE Healthcare) with X-Omat AR film (Eastman Kodak Co., New York, NY, USA), and band intensities were analyzed using LAS-4000 luminescent image (FujiFilm, Tokyo, Japan) as previously described [15 (link)]. C1q expression was normalized to β-actin expression.

Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 1% Triton X-100) supplemented with protease inhibitor cocktail (Complete, Roche Applied Science, Penzberg, Germany). Protein concentrations were determined with Coomassie brilliant blue (Thermo Scientific, Darmstadt, Germany). Protein samples (10–25 µg of total protein per lane) were separated by SDS-PAGE and transferred to Hybond-P polyvinylidene fluoride membranes (GE Healthcare, Boston, MA, USA). After blocking in 5% nonfat dry milk in TBS-T (0.1% Tween 20) for 1 h immunoblots were probed with primary antibodies (Table S5) over night at 4 °C followed by an incubation of peroxidase-conjugated secondary antibody (GE Healthcare, Boston, MA, USA) for 1 h according to the manufacturer’s protocol Blots were detected using WesternBright Chemiluminescence Substrate (Biozym, Hessisch Oldendorf, Germany) and the digital imaging system from Intas (Göttingen, Germany). All Western blot images have been cropped for improved clarity and conciseness. Quantification by densitometry was performed using Image J software (National Institutes of Health, Bethesda, Rockville, MD, USA). Relative band intensities were expressed as arbitrary units and normalized to the corresponding actin.