Bca protein assay

Collected cell pellets were resuspended and briefly sonicated in a cell lysis buffer (Biyuntian Biotechnology Co., Shanghai, CN) containing 1% protease inhibitor cocktail (Biyuntian Biotechnology Co., Shanghai, CN). Cell lysate was spun at 10 000 rpm for 10 min at 4°C, and the resulting supernatant was stored at -80°C. Protein concentration was determined using the BCA protein assay (Biyuntian Biotechnology Co., Shanghai, CN).

Fetal bovine serum and pancreatin were purchased from Gibco. RIPA lysis buffer was purchased from Solabel Technology Co., Ltd. (Beijing, China). BCA protein assay and Tris-Tricine-SDS-PAGE loading buffer were purchased from Biyuntian Biotechnology Co., Ltd. (Shanghai, China). A MTT assay kit was purchased from BioFroxx (Shanghai, China). The annexin V-FITC/propidium iodide double staining apoptosis detection kit was purchased from Kaiji Biotechnology Co., Ltd. (Nanjing, China). CD44v6 antibody was purchased from Abcam. oHA-4 (a hyaluronic acid including four saccharide residues) was purchased from Creativepegworks. 4-methylumbelliferone (4-MU) was obtained from MedChemExpress. BS3 was purchased from Thermo Fisher Scientific. A human hyaluronic acid enzyme-linked immunosorbent assay(ELISA) kit was purchased from Jiancheng Co., Ltd. (Nanjing, China). Oxaliplatin was obtained from Meilun Biotechnology Co., Ltd. (Dalian, China). 1,2-Dilauroyl-sn-Glycero-3-Phosphoethanolamine (DLPE) and 1,2-Dilauroyl-sn-Glycero-3-Glycerol (DLPG) were purchased from Avanti (Alabaster, AL, USA). BALB/c male nude mice were purchased from Sbefu Biotechnology Co., Ltd. (Beijing, China).

The quantitative GUS activity was measured using the Lu’s methods with slight modification [53 (link)]. GUS activity was detected in 1-month-old Arabidopsis leaf tissues (10 mg) from three independent transgenic lines and six individuals in each line. Total proteins were extracted using 300 µL GUS extraction buffer (50 mM phosphate buffer, pH 7.0; 10 mM EDTA, pH 8.0; 0.1 % Sodium Dodecvl Sulfate; 10 mM β-mercaptoethanol). BCA Protein Assay (Beyotime Biotechnology, China) was used to measure the protein concentrations. Extraction (100 µL) was added to 900 µL GUS extraction buffer containing 1 mM 4-methylumbelliferyl glucuronide (MUG, Sigma) and incubated at 37 °C. The 900 µL stop solution (1 M Sodium Carbonate) immediately added into 100 µL the above reaction mixture and 60 min later, respectively. Fluorescence of 4-methylumbelliferone (MU) was monitored using Tecan Infnite™ at 455 nm emission and 365 nm excitation. GUS activity was expressed as µmoles 4-methylumbelliferone (MU) min^− 1 mg^− 1 protein.

The cells were collected in RIPA buffer containing protease inhibitor cocktail (Beyotime, Haimen, China). Proteins were determined using the BCA Protein Assay (Beyotime) and denatured by heat. Equal amounts (20 μg) of protein in loading buffer were added into each well of the SDS‐PAGE gel, separated by electrophoresis, transferred onto PVDF membranes. After blocking by 5% skim milk in TBST (TBS + Tween 20) for about 1 hour at room temperature, the membranes were incubated with primary antibodies against P62 (catalog no. #5114), LC3B (catalog #2775), Bax (catalog #5023), Bcl‐2 (catalog #3498), p‐PI3K (catalog #4228), p‐Akt (catalog #4085), and GAPDH (catalog #5174) at 4°C overnight. All the antibodies were obtained from Cell Signaling (MA). After washed three times with TBST, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (catalog no. sc‐2030; Santa Cruz, CA) for 1 hour at room temperature. Finally, the immunoreaction was detected with an ECL detection kit (Amersham, GE Healthcare).

Protein lysates were generated using RIPA buffer with PMSF (P0013B, Beyotime, Shanghai, China) and protein concentrations were quantified by BCA Protein Assay (P0011/P0012, Beyotime). Lysates were loaded on SDS-PAGE gels for transfer to a PVDF membrane, followed by incubation with the primary antibodies (Table S3) and then with HRP-labeled rabbit anti-mouse IgG (ab6728, Abcam, Cambridge, UK; 1:2000-1:10000) or HRP-labeled goat anti-rabbit IgG (ab6721, Abcam, 1:5000); GAPDH (ab181602, Abcam,1:10000) served as the internal reference. Enhanced chemiluminescence (P0018M, Beyotime) was added, and an ImageQuantLAS4000C gel Imager (GE, USA) was used for development (Li et al. 2018 (link)).

Animals were euthanized with CO₂ at 4, 8, and 12 weeks postoperation, after which eyeballs were enucleated and retinas were quickly isolated on ice. After being rinsed in 0.01 M PBS and drained, retina tissues were lysed in ice-cold tissue lysis buffer [10% phenylmethylsulfonyl fluoride (PMSF) + 90% radioimmunoprecipitation assay (RIPA)]. The lysates were then centrifuged at 15,000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA Protein Assay (Beyotime). After boiling in loading buffer for 10 min, total proteins (10 μg per slot) were electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes. After being blocked in 5% fat-free milk for 2 h at 37°C, membranes were incubated with anti-GFAP antibody (1:500, rabbit, Abcam) and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (1:1,000, mouse, Proteintech Group) antibody overnight at 4°C. Membranes were then incubated with peroxidase-conjugated immunoglobulin G (1:2,000; Santa Cruz Biotechnology). After being washed in Tris-buffered saline with Tween-20 (TBS-T) and developed in developing solution, membranes were scanned using the Bio-Rad exploding system (Bio-Rad, CA, United States) with corresponding software.

Transfected or infected cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer at 4°C for 30 min. The protein concentration was quantified using a BCA protein assay (Beyotime Biotechnology, Shanghai, PRC). Then, the protein lysates were centrifuged at 13,000 × g for 15 min at 4°C to remove the precipitate. The supernatants were collected and added appropriate amount of 6 × SDS loading buffer, and boiled at 100°C for 15 min. Samples were run on SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked with 5% nonfat milk at room temperature for 45 min, and then incubated with primary antibodies and secondary antibodies at room temperature for 1.5 h and 45 min, respectively. After that, horseradish peroxidase substrate was added to the membrane and used for signal detection.