Ice cold lysis buffer

L02 cells were pretreated and incubated as described above. Total cell proteins, nuclear proteins and cytoplasmic proteins from L02 cells pretreated with Hyp were extracted with ice-cold lysis buffer (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instruction. Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA). Protein (30 μg) from each sample was separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% BSA for one hour at room temperature and incubated overnight at 4°C using 1:1000 dilutions of relevant primary mouse antibodies, followed by 1:1000 dilutions of goat anti-mouse secondary antibodies. The protein bands were visualized using the ECL system (Thermo Scientific, Rockford, IL, USA), and the density was determined by Image J software (NIH, USA).

Cardiac fibroblasts were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) to obtain total protein. Protein samples (20 µg) were quantified using BCA Protein Assay kit, and separated by 10% SDS-PAGE, and then the proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore). Then, the membranes were blocked with 5% milk in Tris-buffered saline, followed by incubation with the appropriate primary antibodies: Smad7 (1:1,000; product code ab90086; Abcam); Smad3 (1:1,000; product no. 9513); p-Smad3 (1:1,000; product no. 9520); and β-actin, (1:2,000; product no. 4970; all from CST) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000 dilution; cat. no. 7074; from Cell Signaling Technology, Danvers, MA, USA), and developed using High Sensitivity ECL Substrate kit (product code ab133406; Abcam). The stained protein bands were visualized using Bio-Rad ChemiDoc XRS equipment, and quantified and analyzed using Quantity One software. Three repeats were performed.

Cells were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and centrifugation was performed at 12,000 × g at 4°C for 10 min. Protein concentration was determined using a bicinchoninic acid protein acid kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Proteins (60 µg) were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). The PVDF membrane was incubated with phosphate-buffered saline (PBS) containing 5% non-fat milk (Mengniu Dairy Co., Ltd., Hong Kong, China) overnight at 4°C, followed by incubation with rabbit anti-human FOXC1 antibody (1:50, ab24067) and GAPDH antibodies (1:100, ab9485) (both from Abcam, Cambridge, MA, USA) at room temperature for 3 h. Following 3 washes with PBS with Tween-20, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000, ab97051; Abcam) at room temperature for 1 h. An enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.) was used to detect the immunoreactive bands. Protein expression was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). GAPDH was used as an internal reference. Three replicates were performed.

After administration of fullerenol or normal ACSF for 20 minutes, the hippocampal tissues were removed from the slices and crushed with a pestle in an ice-cold lysis buffer (Beyotime) with protein inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) for 45 minutes. The homogenates were centrifuged at 12,000 rpm for 25 minutes at 4°C. The protein concentrations were determined using a BCA protein assay kit (Beyotime). The protein was mixed with sodium dodecyl sulfate sample buffer, heated to 100°C for 15 minutes, separated under reducing conditions on an 8% sodium dodecyl sulfate–polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was incubated with antibodies against NOS (Boster, Wuhan, People’s Republic of China), CaMKII (Santa Cruz Biotechnology Inc., Dallas, TX, USA), p-CaMKII (Promega, Madison, WI, USA), GAPDH (EMD Millipore), and tubulin (Beyotime) overnight at 4°C with horseradish peroxidase-linked secondary antibody (Promega) for 1 hour at room temperature. The bands were detected using chemiluminescence (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The optical density of each band was determined using ImageJ software and normalized to that of GAPDH or tubulin.