Horse anti mouse igg

The protein concentration was determined using a BCA Kit (Pierce Biotechnology, Rockford, IL, USA). Each protein extract (100 μg) was electrophoresed on a 12.5% SDS-polyacrylamide gel, transferred to PVDF membranes in a buffer containing 25 mMTris-HCl (pH 8.3), 192 mM glycine and 20% (v/v) methanol, and blocked in 5% (w/v) skimmed milk in Tris buffered saline-Tween 20 (0.1% by volume, TBST) for 2 h at room temperature, and probed with specific primary antibodies overnight at 4°C. Blots were reacted with secondary antibodies coupled to horseradish peroxidase in TBST. The primary antibodies were mouse anti-UBE2C (1:1,000; Cat. AM1831a, ABGENT, San Diego, CA, USA), anti-ERK (Cat. 4695S, Cell Signaling Biotechnology), p-ERK (1:1,000; Cat. 9284, Cell Signaling Biotechnology) and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:5,000, Sigma) antibody. The horseradish peroxidase-conjugated secondary antibodies were goat anti-rabbit IgG (1:2,000; Cat. 7,074, Cell Signaling Biotechnology) or horse anti-mouse IgG (1:2,000; Cat. 7,074, Cell Signaling Biotechnology). Signals were detected by a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) according to the manufacturer' s instructions.

ELISA plates (96-well) were coated with either Ad5 (1 × 10⁸ viral particles/well) or purified DE1scFv-pSia (5 µg/well). Plates were then incubated with either serum from blood of Ad5-naive mice, or Ad5-immunized mice, or serum of Ad5-immunized mice pretreated with DE1scFv-pSia to inactivate DE1-specific antibodies as indicated in the figure legend. IgG binding was detected using the following HRP-conjugated secondary antibodies: horse anti-mouse IgG from Cell Signaling Technology (polyclonal; #7076), rat anti-mouse IgG1 from Invitrogen (clone: LO-MG1-2), and rat anti-mouse IgG2a from Invitrogen (clone: LO-MG2a-3). Plates were developed using the BD OptEIA ELISA Kit. The reaction was terminated by addition of 1 mol/l H₃PO₄, and absorbance was measured at 450 nm.

Western blots were performed as previously described [6 (link)]. Proteins transferred to nitrocellulose membranes (Amersham) were immunodetected with mouse anti-Vinculin Antibody (7F9) (sc-73614, Santa Cruz Biotechnology), rabbit anti-mTOR antibody (ab2732, Abcam), mouse anti-Raptor antibody (10E10) (sc-81537, Santa Cruz Biotechnology), rabbit anti-Rictor antibody (53A2) (2114, Cell Signaling Technology). Antibodies used for phosphorylation analysis: rabbit anti-mTOR Ser2448 (#2971, Cell Signaling Technology), rabbit anti-AKT Ser473 (#9271 T, Cell Signaling Technology), rabbit anti-mTOR (#2972 S, Cell Signaling Technology), rabbit anti-AKT (#9272 S, Cell Signaling Technology). Secondary antibodies horse anti-mouse IgG (7076, Cell Signaling Technology) and goat anti-rabbit IgG (7074, Cell Signaling Technology) coupled to HRP were used.

Western blotting was performed as previously described [26 ]. Immediately after enucleation, retinas were dissected and lysed in 1X Mg2+ lysis buffer (EMD Millipore) with a protease inhibitor (Sigma Aldrich, St. Louis, MO) and a phosphatase inhibitor (EMD Millipore). Samples were quantified with Bradford Reagent (Bio-Rad) [27 (link)]. 40 μg of total protein were separated on 4–20% Tris-glycine gel (Bio-Rad), transferred to nitrocellulose membrane (Bio-Rad), and blocked with 5% BSA diluted in Tris-buffered saline supplemented with Tween-20 (TBST) for one hour. Membranes were incubated with rabbit anti-NOX4 (1:200 dilution, sc-30141, Santa Cruz, Dallas, TX) for 18 hours or mouse anti-β-actin (1:5000 dilution, A5316, Sigma-Aldrich) diluted in 5% BSA in TBST for one hour. After washing, membranes were incubated with donkey anti-rabbit (1:5000 dilution, NA934, GE Healthcare, Piscataway, NJ) or horse anti-mouse IgG (1:5000 dilution, 7076, Cell Signaling, Boston, MA) and exposed with ECL Detection Reagents (GE Healthcare). Bands were analyzed using ChemiDoc XRS (Bio-Rad) and normalized to β-actin. Data are fold change ± SEM.

Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (PIC, 1x), sodium fluoride (NaF, 200 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM), ethylenediaminetetraacetic acid (EDTA, 5 mM) and calyculin A (50 nM, Cell Signaling, USA). For the preparation of protein samples from CM, PIC (1x) and EDTA (5 mM) were added into the cell culture supernatant fraction. Following the quantitation with colorimetric bicinchoninic acid (BCA) assay, total protein (50–120 μg) was subjected to SDS-PAGE on 10% gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk powder in PBS-T buffer and incubated overnight at 4 °C with primary antibodies reactive with human phospho-STAT3 Tyr705 (pSTAT3; D3A7, 1/1500), total STAT3 (tSTAT3; 79D7, 1/1500), and β-actin (D6A8, 1/5000) (Cell Signaling), fibronectin (10/Fibronectin, 1/1000) (BD), FN-EDA (IST-9, 1/1000) (Abcam, UK). Following the incubation with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, 1/3000; horse anti-mouse IgG, 1/1500) (Cell Signaling) (1.5 h, at room temperature), and then, with ECL substrate solution, protein bands were visualized and densitometric analyses were performed (Kodak Gel Logic 1500, Carestream, USA). Otherwise stated, all materials were purchased from Thermo Scientific, USA.