Ec3 imaging system

Small pieces of frozen tissues were placed into 1.5 mL centrifuge tubes and then homogenized by using a Turrax homogenizer (IKA, Germany) in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orhtovanadate, 0.5% Triton X-100, 1mM PMSF, protease inhibitor cocktail). Tissues lysates were obtained by centrifugation at 14.000g for 20 min at 4°C.
Equal amount of protein were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride transfer membrane (Immobilon-P transfer membrane, Millipore, MA) using a Trans-Blot semi-dry transfer cell (BioRad, CA, USA). Blots were incubated with the appropriate antibody (anti-SERPINE2 1:1000, R&D Systems, MN, USA; anti-ITIH5 1:1000, ABCAM, UK; anti-WISP2 1:1000, Abnova, Taiwan). Immunoblots were visualized with Immobilon Western Detection kit (Millipore, MA) using horseradish peroxidase-labeled secondary antibody. To confirm equal loading for each sample, membranes were incubated with stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-β-actin antibody (Sigma, MO, USA). Images were captured and analyzed with an EC3 imaging system (UVP, CA, USA).

VEGF protein levels were detected by Western blot, using 30 μg of protein in 8.5% SDS-PAGE electrophoresis gel and electroblotted. The membranes were incubated with mouse anti-VEGF antibody (1:1000, ThermoScientific) overnight. Next, the membranes were incubated with a secondary antibody and HRP-conjugated anti-mouse IgG (1:5000, Santa Cruz Biotechnology). The proteins were detected with an enhanced chemiluminescence kit (Millipore) and by radiography. All Western blot analyses were performed within the linear range of protein loads and antibody use. The bands were scanned for densitometric analysis using the UVP EC3 Imaging System and the UVP VisionWorks LS Image acquisition and Analysis Software.

DNA diffusion degradation and fragmentation of HCECs was detected by agarose gel electrophoresis (Fan and Fan, 2017 (link)). HCECs were cultured in 25 cm² flasks at a density of 1 × 10⁶ cells/5 mL/flask, and harvested as mentioned. After being washed three times by ice-cold PBS and centrifuged (1000 g, 15 min), their total genomic DNA was extracted with a TIANamp Genomic DNA Kit (Cat No:DP304, TianGen Biotechnology, Beijing, China). All DNA samples from each group were electrophoresed in a 1% agarose gel (200 mA, 260 min), the gel was stained with 0.5 μg/mL EB solution for 10 min, and observed with an UVP EC3 imaging system (UVP, Upland, CA, United States).

For reduced and denatured conditions, protein samples were resolved on NuPAGE Novex Bis-Tris Gels (Life Technologies) using the MOPS/LDS buffer system or Mini-PROTEAN TGX Precast Gels (Bio-Rad) using the Tris/Glycine/SDS buffer. Native protein samples were resolved on 4–16% NativePAGE Novex Gels (Life Technologies) using 0.001% G-250 cathode buffer. Proteins were transferred to PVDF membranes and incubated with primary antibodies overnight (see following table for antibody information) and detected with either anti-rabbit or anti-mouse (GE Healthcare), or anti-goat (Sigma) HRP-conjugated antibodies and visualized with ECL Advance substrate (GE Healthcare) using the EC3 Imaging System (UVP). For quantification of relative protein levels, densitometry analysis was performed using LI-COR Image Studio Lite. For blue native PAGE analysis of mouse tissues, 50 mg wet weight of tissue was homogenized for 7 min at 50 Hz using the TissueLyzer II (Qiagen) in Native PAGE sample buffer (Invitrogen) with Halt protease and phosphatase inhibitors (Thermo) and 1% digitonin. Lysates were cleared at 20,000 × g for 30 min at 4 °C. G-250 Sample Additive (Invitrogen) was added to the samples and blue native PAGE was run using the dark blue and light blue cathode buffer protocol per the manufactures instructions (Invitrogen). Proteins were transferred and blotted as described above.

The sciatic nerve tissues were subjected to quantitative RT-PCR experiments. Total RNA was isolated from maxilla tissues by using Trizol reagent in accordance with the manufacturer’s protocol. The extracted RNA (1 μg) was reverse-transcribed into cDNA by using a PrimeScript RT reagent kit. RT-PCR was performed and from each PCR reaction. The primer sequences of NF-κB, Nrf2, and GAPDH were designed as follows: NF-κB p65, FW: 5′-GACGAGGCTCGGAGAGCCCA-3′ and RV: 5′-CTGGGGCGGCTGACCGAATG-3′; Nrf2, FW: 5′-TCCATTTCCGAGTCACTGAACCCA-3′ and RV: 5′-TGACTCTGACTCCGGCATTTCACT-3′; GAPDH, FW: 5′-CCTGGAGAAACCTGCCAAG-3′ and RV: 5′-CACAGGAGACAACCTGGTCC-3′. After PCR was completed, the products were analyzed through electrophoresis on 1.2% agarose gel and photographed under UV light in an EC3 Imaging System (UVP, Upland, CA).

After the cell treatment, cells were rapidly washed with ice-cold phosphate buffered saline and scraped in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM Sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% Triton X-100, 1 mM PMSF and protease inhibitor cocktail from Thermo Scientific). Lysed cells were centrifuged at 14.000 g for 20 min. SDS-PAGE and blotting procedure were carried on as previously described38 (link). Immunoblots were incubated with the appropriate antibody (anti-PGRN diluted 1:1000, Santa Cruz, CA, USA; anti-NOS2 diluted 1:1000, Cell Signalling, MA, USA; anti-COX2 diluted 1:50, anti-VCAM-1 diluted 1:1000, Cell Signalling, MA, USA; anti-MMP13 diluted 1:500, Santa Cruz, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore, MA) using anti-rabbit (GE Healthcare, UK) horseradish-peroxidise-labelled secondary antibody diluted 1:2000. To confirm equal loading in each sample, the membranes were stripped in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-GAPDH antibody diluted 1:30000 (Sigma, MO, USA). The images were captured and analyzed with an EC3 imaging system (UVP). Densitrometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Western blotting was performed as previously described.22 Briefly, the polyvinylidene difluoride (PVDF) membrane by Millipore (USA) was used to incubate with the first antibody against AKT (Mouse, 1:1000) (#2920), phospho‐AKT (Ser473) (Rabbit, 1:1000) (#4060),Actin (Rabbit, 1:2000) (#8457) from Cell Signaling Technology (MA, USA), NLGN3 (Mouse, 1:500) (sc‐137052) that was from Santa Cruz Biotechnology. The recombinant protein neuroligin 3 was obtained from Novus biologicals (9069‐NL). The first antibodies were labeled with a secondary antibody, protein bands were imaged using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). The EC3 Imaging System (UVP, LLC, Uplant, USA) was used to obtained blot images directly from the PVDF membrane. The data of Western blot were quantified using Image J Pro Plus 6.0 software.