Sc 20357

Rat knee joint tissues were homogenized and lysed in a protein extraction reagent (Tissue protein extraction reagent, Pierce, Rockford, US). Western blot analysis was performed as described previously [29 (link)]. The primary antibodies to transferrin (17435-1-AP; Proteintech), catalase (ab209211; Abcam), SOD1 (ab16831; Abcam), SOD2 (ab68155; Abcam), SOD3 (ab21974; Abcam), glutathione peroxidase 1 (ab59546; Abcam), glutathione peroxidase 2 (MAB5470; R&D Systems), transthyretin (ab9015; Abcam), NF-κB (8242S; Cell Signaling), IL-1β (ab9722; Abcam), RANKL (sc-9073; Santa Cruz), and GAPDH (sc-20357; Santa Cruz) were obtained commercially. After extensive washing, appropriate secondary antibodies were used in the experiments, including anti-rabbit IgG antibody conjugated with horseradish peroxidase (7074S; Cell Signaling), anti-mouse IgG antibody conjugated with horseradish peroxidase (7076P2; Cell Signaling), or anti-sheep IgG antibody conjugated with horseradish peroxidase (313-035-003; Jackson ImmunoResearch).

Cells were treated with 40 μM of RSV with or without ALA for 24 h. For protein analysis, whole cell protein lysates from HepG2 were harvested with RIPA buffer (50 mmol/l Tris, 150 mmol/l NaCl, 0.5% sodium deoxycholate (v/v), 0.1% SDS (w/v), 1% NP-40 (v/v), pH 7.4) supplemented with proteinase inhibitors. Protein concentrations were determined with the Pierce™ BCA assay (Thermo Fisher Scientific GmbH, life technologies™, Darmstadt, Germany) according to the manufacturer's instructions and analyzed by Western Blotting analysis as described before (40 (link)). Briefly, 30 μg per samples were heated with loading buffer and applied to Mini-PROTEAN® Stainfree™ Precast Gels (4–20%, BioRad laboratories GmbH, Munich, Germany). Samples were transferred to a polyvinylidenedifluoride membrane (Bio-Rad laboratories GmbH, Munich, Germany) and blocked with skim milk dissolved in Tris-buffered saline + 0.05% (v/v) Tween 20. Membranes were then incubated with the primary antibodies FADS1, FADS2, CPT1, GAPDH (sc-134337, sc-98480, sc-48357, and sc-20357, respectively; Santa Cruz Biotechnology Inc., Dallas, USA) and FASN (bs-5045R, Bioss Inc., Massachusetts, USA). Secondary antibodies were from Santa Cruz Biotechnology Inc., Dallas, USA. Results were analyzed using Image Lab 5.0 (Bio-Rad laboratories GmbH, Munich, Germany).

Proteins from breast tumors were extracted with buffer A (50 mM Tris pH = 6.8, 2% SDS, 5% glycerol, 2 mM DTT, 2.5 mM EDTA, 2.5 mM EGTA, 4 mM sodium orthovanadate, 20 mM sodium fluoride, 1 mM PMSF). The antibodies used in this study were: anti-FOXO3 (9467, Cell signalling, Beverly, MA, USA), anti-Phospho-FOXO3A (pSer-253) (ab47285, abcam, Cambridge, MA), and anti-GAPDH used as internal control (sc-20357, Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected by the ECL Western Blotting Analysis System procedure (GE Healthcare, Buckinghamshire, UK).

The total proteins were extracted, and the protein concentration was measured as described previously [8 (link)]. Total proteins (10 μg/lane) were separated by SDS-PAGE followed by transferring onto 0.22 μm PVDF membranes (Millipore, Bedford, MA, USA). Blot analysis was performed with an anti-IFN-γ antibody (Abcam, San Francisco, CA, USA, ab27919), an anti-TNF-β antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, SC-28345), an anti-IL-2 antibody (Abcam, ab193807), an anti-IL-4 antibody (Bio-Techne, MAB2469), an anti-IL-5 antibody (Santa Cruz Biotechnology, SC-8433), an anti-IL-6 antibody (Abcam, ab193853), and an anti-IL-10 antibody (Santa Cruz Biotechnology, SC-32815) at a dilution of 1:1000 to analyze expression of Th1 and Th2 cytokines respectively. Target proteins were detected by a Pro-light HRP chemiluminescence detection reagent (Tiangen Biotech, Beijing, China). An anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, sc-20357, 1:1000) was used for normalization of sample loading. The intensity of blots were semi-quantified by Quantity One V452 (Bio-Rad Laboratories), and the values were calculated using GAPDH as an internal reference.

An SDS-page was performed using 25 μg of total protein loaded into a 10% polyacrylamide gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane using a semi-dry electroblotter (Bio-Rad). The membrane was processed for chemiluminescence detection using Luminata Forte Western HRP Substrate (Millipore) according to the manufacturer's instructions. The primary antibodies were: rabbit anti-TRPM8 (Ab109308, Abcam, detecting the pore region of TRPM8), rabbit anti-HA tag (Sc-805, Santa Cruz) and goat anti-GA3PDH (Sc-20357, Santa Cruz).

iPS cells and neurospheres were isolated, suspended in RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich), triturated and centrifuged at 10,000g, and 4°C for 10 min. The supernatants were separated on 10% SDS–PAGE gels (10 μg protein/lane) and blotted onto a polyvinylidene difluoride membrane. The membranes were then blocked with 5% skim milk, incubated with anti-AGE antibodies (CML: TransGenic, KH001, dilution 1/500; CEL: COSMO BIO, AGE-M02, dilution 1/1,000; MG-H1: CELL BIOLABS, STA-011, dilution 1/1,000; PEN: TransGenic, KH012, dilution 1/500; ARP: NOF CORPORATION, 5F6, dilution 1/500), or anti-GAPDH antibody (sc-20357, dilution 1/1,000; Santa Cruz Biotechnology) at 4°C overnight and then incubated with HRP-conjugated IgG (dilution 1/5,000; GE Healthcare) for 1 h at room temperature. The resulting signals were detected with an Immobilon Western Chemiluminescent HRP Substrate (Merck MilliporeSigma), and the bands were analyzed using a FUSION Solo instrument (Vilber Lourmat). The intensities of the bands were quantified using Fusion software (Vilber Lourmat). The expression level of GLO1 was normalized to that of GAPDH.