Pvdf blotting membrane

The proteins from RPE cells were lysed using RIPA buffer and centrifuged at 12 000 ×g for 20 minutes, and protein concentration was determined by the Bio‐Rad protein assay kit (Bio‐Rad). Proteins were resolved on Tris‐HCl 4‐12% polyacrylamide gels at 110 V. The proteins were transferred to PVDF blotting membrane (Millipore, Bedford, MA). The membranes were blocked in 5% non‐fat milk and probed with antibody specific for Phosph‐Smad2/3 (Abcam), α‐SMA (Sigma‐Aldrich), Collagen1 (Biosis), FN (Abcam), PPAR‐γ (Biosis), P‐MeCP2‐421,80 and total MeCP2 overnight at 4°C. Membranes were washed and incubated with a horseradish peroxidase–conjugated secondary antibody (Vector Laboratories) for 30 minutes at room temperature. Images were visualized by addition of ECL chemiluminescence detection solution (Amersham Pharmacia Biotech). After stripping, the membranes were re‐probed with anti‐GAPDH antibody (Millipore) for protein loading control (Li, et al)²⁷.

Total protein extraction from macrophages plated in 6-well plates was performed using a lysis buffer containing 40 mM Tris pH 7.5, 150 mM KCl, 1 mM EDTA, 1% Triton-X-100, PIC (Protease Inhibitor Cocktail, Roche), PIB 25x (Phosphatase Inhibitor Buffer, 25 mM Na₃VO₄, 250 mM PNPP, 250 mM β-glycerophosphate, 125 mM NaF). Cell lysate was recovered and centrifuged for 5 min at 15,700 g and 4 °C to pellet cell debris. The supernatant was collected and stored at −70 °C before western blotting. 20 µg of proteins were separated on 10% SDS-PAGE gels and transferred onto a low fluorescence background PVDF blotting membrane (Millipore). Quantitative LI-COR technology was used for western blot analyses (Odyssey Infrared Imaging System v3.0.16, LI-COR, Biosciences). Membranes were blocked with Odyssey blocking buffer diluted 1:2 in PBS for 1 h at RT. Primary antibodies diluted in Odyssey Blocking buffer-Tween 0.1% were incubated overnight at 4 °C, then membranes were washed with PBS-Tween 0.1%, and finally incubated with secondary antibodies diluted 10,000 x for 1 h at RT. Membranes were washed with PBS-Tween 0.1%, and then with PBS, and finally dried before scanning. Loading control was assessed with α-tubulin or β-actin according to the molecular weight of the protein of interest. Antibodies used are listed in Supplementary Table S2.

HRMECs were counted and separated into three groups: a negative control; a high glucose damage model treated with safranal (10 μL, 80 g/mL); and a high glucose damage model treated with high glucose (10 μL, 25 mol/L). A Bio-Rad test kit was used to extract the cells’ total protein and evaluate the protein content (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (30 μg) were resolved on polyacrylamide gels containing 12% Tris-HCl before being transferred to a PVDF blotting membrane (Millipore, Billerica, MA, USA). After blocking, specific antibodies against phosphate-extracellular regulated protein kinases (p-ERK), total-ERK (t-ERK), p-P38, t-P38, p-serine/threonine kinase (AKT), t-AKT (1:2000, Santa Cruz, CA, USA), E-cadherin, N-cadherin, Snail, Twist, Fibronectin(1:2000, Santa Cruz, CA, USA) and beta actin (1:2,000, Abcam, Cambridge, MA, USA), were treated with the membranes. The protein bands were detected by chemiluminescence after the blots had been carefully washed and treated with peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (1:1,000, ZSGB-Bio, Beijing, China) (Pierce, Rockford, IL, USA). Three times the experiment was conducted (10 (link)).

Cells were lysed, supernatants were collected, and proteins were resolved on Tris-HCl 10% polyacrylamide gels (Ready Gel; Bio-Rad, Hercules, CA) at 120 V. The proteins were transferred to PVDF blotting membrane (Millipore, Bedford, MA). The membranes were probed with antibody for phospho-αB crystallin (Ser 59, Stressgen), pan-AKT (C67E4, Cell Signaling), phospho-AKT (Ser 473, Cell Signaling), phospho-PDK1 (Ser 241, Cell Signaling), phospho-c-Raf (Ser 259, Cell signaling), phospho-GSK-3β (Ser 9, Cell Signaling), PPAR_γ (Santa Cruz Biotechnology) all at 1∶1,000 dilution. Membranes were washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1∶3,000, Vector Laboratories, Burlingame, CA) for 30 min at room temperature. Images were developed by adding ECL chemiluminescence detection solution (Amersham Pharmacia Biotech, Cleveland, OH). Monoclonal anti mouse GAPDH was used as the loading control.

Protein was extracted from cells and posterior eyecup without conjunctiva, sclera, and muscle tissue with RIPA buffer containing protease inhibitor. The concentration of soluble protein was measured using BSA as standard (SpectraMax iD5, Molecular Devices, Sunnyvale, CA, USA). Equal amounts of protein (30 μg) were resolved on TGX-precast gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to PVDF blotting membranes (Millipore, Billerica, MA, USA). Membranes were probed with respective primary antibodies overnight at 4 °C (The antibodies and dilutions used are listed in Table 2). After incubation with the appropriate secondary antibodies (Vector Laboratories, Burlingame, CA, USA), protein bands were visualized by a chemiluminescence (ECL) detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal protein loading was confirmed with GAPDH. Protein band intensity was measured by Image J software (Version 1.51 23 April 2018 and URL accessed on 12 April 2018) (https://imagej.nih.gov/ij/). The quantification indicates the relative amounts as a ratio of each protein band to the appropriate loading control and expressed arbitrary units [35 (link)].

Cells grown to confluence on 6-well culture plates (VWR) were harvested after the specified treatment period and washed with PBS. Protein was extracted from the cells using mammalian protein extraction reagent with protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL), and quantified with a protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved on 8–16% Tris-HCL polyacrylamide gels (Pierce Biotechnology, Rockford, IL) and transferred to PVDF blotting membranes (Millipore, Billerica, MA). Membranes were probed with rabbit polyclonal anti-gamma glutamyl cysteine ligase, catalytic subunit (GCLC) (Abcam, Cambridge, MA), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, MA), mouse anti-CHOP (Pierce Biotechnology, IL), rabbit anti-GRP78 (Sigma Aldrich, St. Louis, MO), rabbit anti-COX IV (Cell Signaling Technology, MA), rabbit polyclonal anti-GRX2 (GeneTex, Irvine, CA) and rabbit anti-β-Tubulin (Cell Signaling Technology, MA) overnight at 4°C. After incubation with corresponding secondary antibody tagged with horseradish peroxidase, signals were detected using an ECL chemiluminescence system (Pierce Biotechnology, IL). Membranes were then stripped and reprobed with monoclonal anti-GAPDH (Millipore, Billerica, MA). Protein band intensity was measured by Image Studio Software (Li-Cor, Lincoln, NE) [30 (link)].