Protein extraction buffer

The remaining SHR (n = 5) were deep narcotized in each group and perfused intracardially with 50 mL ice-cold 0.9% saline. Then the entorhinal cortex of brain tissue (from Bregma −4.80 to −7.04) was rapidly dissected and homogenized in protein extraction buffer (Thermo, Pierce Biotechnology, Waltham, MA, USA) containing a complete protease inhibitor cocktail (Thermo). Protein concentration was measured with the Bradford Protein Assay (Thermo). Equal amounts of the protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat dried milk for 1 h at room temperature, the membranes were incubated with rabbit anti-nestin (1:200; Millipore), mouse anti-synaptophysin (1:100, Abcam) rabbit anti-Bcl-2 (1:1000; Cell Signaling Technology) and mouse anti-β-actin (1:1000; Santa Cruz) overnight at 4°C. The membranes were then incubated with the appropriate secondary antibodies (1:1000; Cell Signaling Technology) for 2 h at room temperature. Positive signals were detected by enhanced chemiluminescence (ECL, Cell Signaling Technology) and visualized by exposure to X-ray film.

Corneal tissues (6 eyeballs/group from two batches) were harvested and homogenized with protein extraction buffer (Thermo Fisher Scientific). The mixture from each sample was then centrifuged, and the supernatant was collected. Total protein of cornea lysate was quantified by Bradford assay (p010, GeneCopoeia, Rockville, MD, USA). An equal amount of total protein (15 µg in 100 µL) from each sample was used to quantify the VEGF and MMP-9 by ELISA (Quantikine ELISA kits, R&D system, Minneapolis, MN, USA). This experiment was conducted according to the manufacturer’s protocol.

Protein extraction and immunoblotting were performed as previously described 30 (link), 31 (link). In brief, cells were lysed in the protein extraction buffer (Thermo Scientific, Rockford, IL, USA). Then, the proteins were collected, loaded on a sodium dodecyl sulfate-polyacrylamide gel, and transferred to nitrocellulose. Immunoblotting was performed using primary and secondary antibodies.

Protein extraction and immunoblotting were performed as previously described [20 (link),29 (link)]. Briefly, cells were lysed in a protein extraction buffer (Thermo Fisher Scientific, Inc.). Next, the proteins were collected, loaded on a sodium dodecyl sulfate–polyacrylamide gel, and transferred to nitrocellulose membranes. Immunoblotting was performed using primary and secondary antibodies. Signals were detected using a chemiluminescence assay and captured using the ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Proteins from HUVECs were isolated with protein extraction buffer (Thermo Fisher) by incubation on ice for 30 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, supernatants were collected. Protein concentrations were determined by a BCA protein assay kit (Thermo Fisher). Western blotting was performed as described previously [20 (link)]. In brief, Total 20 μg of protein samples were loaded at SDS-PAGE electrophoresis gel for running. Then the protein was transferred to NC membrane and blocked in 5% milk powder. Membranes were incubated with specific primary antibodies [anti-BCL2 antibody (Rabbit, 1:1000 dilution), anti-BAX antibody (Rabbit, 1:1000 dilution), anti-HDAC4 antibody (Rabbit, 1:1000 dilution), anti-GAPDH antibody (mouse, 1:1000 dilution) and anti-β-Actin antibody (mouse, 1:1000 dilution), from Cell Signaling Technology] at 4 °C overnight. Then the membranes were washed and incubated with a secondary antibody (Li-COR Biosciences, Lincoln, NE) conjugated with IRD800 at room temperature for 1 h in the dark. The membranes were then washed and analyzed by the Odyssey software system (Li-COR). The ratio of the protein change was normalized to GAPDH.

Proteins were extracted from the samples using a protein extraction buffer (Thermo Scientific, Cat #78510) mixed with 100 × phenylmethylsulfonyl fluoride (PMSF) and a single cOmplete Mini, EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, Basel, Switzerland, Cat #22836170001). The homogenized samples were centrifuged, and the supernatants were collected. The protein concentration was measured using the Bradford assay (Bio-Rad, Cat #5000205). Equal quantities of proteins were loaded onto an SDS-polyacrylamide gel and electrophoretically transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, United States, Cat #1704158). The membranes were treated overnight at 4°C with primary antibodies (refer to Table 3 for the list of antibodies used) and then for 1 h with a horse radish peroxidase-conjugated secondary antibody. The membranes were developed using Amersham Imager 600. The bands were measured using ImageJ program. The bands were selected by drawing a frame around them using the “rectangle” tool. The area was assigned and then profile plot of the lane was acquired using “plot lanes” tool. Each peak in the profile plot represents the relative intensity of the bands in the selected lane. Straight line selection tool was used in order to enclose each peak, then the wand tool was used to quantify the intensity.