Amersham imager 680 blot and gel imager

Proteins were extracted from tissues using lysis buffer (4% SDS; 2 mM TCEP; and 0.1 M Tris–HCl, pH 7.4). Insoluble pellets were removed by centrifugation at 15,000 rpm at 21 °C for 20 min. Lysates containing 10 µg of protein were loaded on SDS-PAGE gel and subsequently blotted onto a PVDF membrane (Millipore, Darmstadt, Germany). After blocking for 2 h with 5% bovine serum albumin (BSA) in TBS-T buffer, the membrane was incubated overnight at 4 °C with one of the following antibodies: anti-beta-actin (1:2000 dilution; sc-47778; Santa Cruz, Dallas, TX, USA), anti-EPCAM (1:1000 dilution; sc-21792; Santa Cruz, Dallas, TX, USA) and anti-P4HB (1:1000 dilution; 2446; Cell Signaling, Danvers, MA, USA). After three washing steps with TBS-T buffer, the membrane was incubated for 2 h with a secondary anti-mouse (1:5000 dilution; 31430; Invitrogen, Carlsbad, CA, USA) or anti-rabbit (1:5000 dilution; 31460; Invitrogen, Carlsbad, CA, USA) antibody and washed again three times with TBS-T buffer. Finally, protein expression on the membranes was measured by a Amersham Imager 680 blot and gel imager (GE, Boston, MA, USA) using a SuperSignal™ West Femto maximum sensitivity substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Expression of the fSPT transgene was detected in total tissue lysates. Sciatic nerves were harvested and homogenized in RIPA buffer (ThermoFisher Scientific) supplemented with protease inhibitor cocktail (ThermoFisher Scientific) and phosphatase inhibitor cocktail (Roche Life Science, Indianapolis, IN). Liver was harvested and homogenized in Cell Lysis Buffer (Cat#9803 s, Cell Signaling Technology, Danvers, MA) with protease inhibitors (Crystalgen, Commack, NY). Samples were incubated 30 min on ice and later spun at 14,000 x g at 4°C for 10 min. Proteins were resolved on a 4–12% Bis-Tris gel (ThermoFisher Scientific), then transferred to nitrocellulose membranes (ThermoFisher Scientific). The membranes were blocked in 5% non-fat dry milk, 0.05% Tween-20 in TBS, then probed with the indicated antibodies. fSPT expression was detected using monoclonal anti-human SPTLC1 (clone H-1, sc-374143, 1:1000 dilution) (Santa Cruz Biotechnology, Dallas, TX), followed by anti-mouse IgG, HRP-conjugated (AP-124P, Millipore, Burlington, MA). Membranes were reprobed with anti-mouse β-actin (monoclonal AC-15, HRP-conjugated) (Abcam, Cambridge, MA) to provide a loading control. Signal was detected by chemiluminescence (ECL Prime Western Blotting Detection Reagents, GE Healthcare Bio-Sciences, Pittsburgh, PA) visualized with an Amersham Imager 680 blot and gel imager (GE Healthcare Bio-Sciences).

A quantity of 7.0 × 10⁵ cells was seeded in a 6 cm dish and the cultured medium with serum-free medium was collected for 40 h. The supernatant of the collected culture medium was centrifuged at 4 °C 12,000× g for 10 min. The 5–10 μg samples were mixed with 5× sample dye (non-reducing), and electrophoresis separation was performed at 150 V (SDS-PAGE with 2% gelatin in the gel, running buffer: Tris Base, glycine, and 0.1% SDS). After washing the gel with washing buffer (containing Triton X-100, Tris-HCl, CaCl₂, and ZnCl₂), the gel was incubated in a 37 °C incubator for 17–24 h with incubation buffer (containing Triton X-100, Tris-HCl, CaCl₂, and ZnCl₂). The gel was stained with staining buffer (containing methanol, acetic acid, and Coomassie Blue) and persistently destained with destain buffer (containing methanol and acetic acid) until the white band was visualized. The band was analyzed using an Amersham Imager 680 blot and gel imager (GE healthcare) and MultiGauge image analysis software version 3.0 (Fujifilm, Stockholm, Sweden).

NPs were loaded with human IL-4, human IL-4/GFP, mouse IL-4 or mouse IL-4/GFP pDNA (Sino Biological, Wayne, PA) based on the molar ratios of PEI nitrogen (N) to DNA phosphate (P) (N/P ratio). The following equation was used to determine the N/P ratio, where the molecular mass (MM) of the repeating unit for branched PEI is 43 Da, the number of primary nitrogen’s in the repeating unit of branched PEI is 4, and the MM of DNA used was 330 Da (30 (link), 31 (link)).
DNA encapsulation by NPs was investigated by agarose gel electrophoresis. NPs were loaded with pDNA at different N/P ratios and ran on a 1% agarose gel for 30 minutes. pDNA was visualized with an Amersham Imager 680 blot and gel imager (GE Healthcare, Chicago, IL).
The IL-4/GFPpDNA-NPs were made at different N/P ratios in PBS. NPs suspension and IL-4pDNA solutions were prepared before each experiment at various N/P ratios. First, 1 μl of NP suspension (including of 0.1 μg of PEI) was mixed with 20 μl of PBS to disperse the NP suspension. Second, the prepared IL-4pDNA solution was added into the NP suspension with various amounts to meet the different N/P ratio. Third, IL-4pDNA solution was well mixed with the NP suspension, then the mixture was incubated for 30 minutes at room temperature before transfection.

Western blotting analysis was performed to evaluate the expression of MMP. The cells were washed twice with cold phosphate buffered saline and lysed. Equal amounts of cell extracts were resolved by 4–20% SDS-PAGE and analyzed by Western blot. The separated proteins were transferred onto nitrocellulose membranes (Hybond-ECL, GE healthcare, Buckinghamshire, UK). In the next step, the membranes were blocked with 4% nonfat milk in Tris buffered saline containing 0.1% Tween 20 at room temperature. Proteins of interest were then detected with the primary antibodies (MMP 1 and 2: Cell signaling, Beverly, MA; MMP9: Santa Cruz Biotechnology, Santa Cruz, CA, USA) and with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) using an enhanced chemiluminescence detection system (Amersham™ ECL™ Select Western Blotting Detection Reagent; GE Healthcare) in accordance with the manufacturer’s instructions. Each blot was probed with an anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Bands were detected using AI 680 (Amersham™ Imager 680 – Blot and Gel Imager, GE Healthcare) and intensity quantification was performed using the ImageJ software (version 1.52; National Institutes of Health, Bethesda, MD, USA). The protein expressions in the treated cells were divided by the level of β-actin to calculate relative protein expression ratios.