Bicinchoninic acid reagent

Immunoblot analysis was conducted by a standard protocol^{47 (link),48 (link)}.In short, kidney cortex tissue and cell lysates were treated with 2% sodium dodecyl sulfate (SDS) buffer containing protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined with the bicinchoninic acid reagent from Thermo Fisher Scientific. The same amounts (100 μg) of protein were loaded in each lane and resolved on SDS-polyacrylamide electrophoresis gel and then transferred to polyvinylidene diflouride membrane which was then blocked with 5% fat-free milk for 1 h before incubation with primary antibodies and horseradish peroxidaseconjugated secondary antibodies. The protein bands were revealed with a chemiluminescence kit (Termo Fisher Scientific). Densitometry of protein band signal was analyzed with Image J software (NIH) for quantification.

Spinal cord tissue and cells were lysed by RIPA with phosphatase inhibitors and protease inhibitors cocktail, and protein concentration was determined using a bicinchoninic acid reagent (ThermoFisher). Equal amount of proteins was separated using 8–12% SDS-PAGE gels, and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Then the membranes were blocked with 5% nonfat milk and incubated with primary antibodies: Mettl14 (1:1000, #51,104, CST, Beverly, MA, USA), MAP2 (1 µg/mL, ab11267, Abcam), acetyl-α-tubulin (0.06 µg/mL, ab24610, Abcam), Iba-1 (1:500, ab178846, Abcam), RASD1 (0.4 µg/mL, NBP1-91830, Shanghai Univ Biotechnology Co., Ltd., Shanghai, China), p-4EBP1 (1:1000, #2855, CST), t-4EBP1 (1:5000, ab32024, Abcam), p-mTOR (1:1000, ab109268, Abcam), t-mTOR (1:1000, ab32028, Abcam), and secondary antibodies (1:2000, ab205718 or 1:2000, ab205719; Abcam). The bands were examined by the ChemiDic XRS + Imaging System (Bio-Rad), and band intensities were analyzed with Image Lab 3.0 software (Bio-Rad). Experiments were performed at least three times.

The level of intracellular ROS was measured using the reagent, 2′,7′-Dichlorofluorescin diacetate (DCFDA; Sigma-Aldrich). After treatment, the cells were washed and incubated for 1 h at 37 °C with DCFDA (5 μM) in culture medium without serum. The cells were washed with PBS and lysed in the lysis buffer (10 mM Tris pH 7.9, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.2 mM Na₃VO₄, 0.5% NP-40 and 1% Triton X-100). After centrifugation, an aliquot (10 μl) of the supernatant was mixed with 90 μl of PBS in a 96-well black plate. Fluorescence was measured using Infinite M200 Pro Multimode Microplate Reader (Tecan) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The ROS readings were normalized with the amount of protein present in each sample. Protein concentration was determined using bicinchoninic acid reagent (Thermo Fisher Scientific) with bovine serum albumin as standard.

The activity of MMP-2 was determined through gelatin zymography based on the visualization of free-gelatin areas digested by MMP-2. Briefly, medium samples were mixed with sample buffer containing 10% SDS in a 1:4 proportion. Electrophoresis of the samples on 10% gel polyacrylamide gel with 0.05% gelatin type A from porcine skin (G2500) (Sigma–Aldrich, USA) was performed to separate the enzyme. The gels were washed for 1 h to remove the SDS and then incubated overnight at 37 °C in a buffer containing 1% Triton X-100 (pH 7.2). Gels were stained using 0.1% Coomassie Blue R-250 in 20% methanol and 10% acetic acid, and subsequently, the stain was removed in 20% methanol and 10% acetic acid. MMP-2 activity was detected as clear bands on a blue background. Enzyme was identified by comparing its localization with standards of gelatinases (R&D Systems Inc., Minneapolis, USA). Zymographic gels were scanned and quantified using ImageJ software (National Institute of Health, Bathesda, USA). The results were expressed as the % activity of secreted MMP-2 versus the non-treated control group (100% of activity) [31 (link)].
The protein concentration in the cell sample medium was determined using the bicinchoninic acid reagent (Thermo Fisher Scientific, Waltham, MA, USA).

hPDLCs (hPDLCs1 and hPDLCs2) were cultured in 60-mm dishes for 1 week, starved for 12 h after reaching 90% confluence, and then treated with APN three times (once every 2 days) during the culture period. Total protein was extracted using RIPA buffer (CW2333S; Applygen, Beijing, China) and the concentration was measured with bicinchoninic acid reagent (Thermo Fisher Scientific, MA, U.S.A.). Protein samples (40 mg/lane) in loading buffer were separated by SDS/PAGE and transferred on to a polyvinylidene difluoride membrane (Millipore, MA, U.S.A.). The primary antibodies used were as follows: anti-GAPDH (g0314; Santa Cruz Biotechnology, California, U.S.A.), anti-runt-related transcription factor 2 (RUNX2) (8486S; Cell Signaling Technology (CST), MA, U.S.A.), anti-P-p38 (4511; CST), anti-p38 (8690; CST), anti-P-p65 (3033; CST), anti-p65 (3034; CST), anti-APPL1 (3858S; CST) at 1:1000. Anti-rabbit secondary antibody (7074S; CST) at 1:5000 was used as described previously [16 (link)]. The Western blots were scanned using an Odyssey® CLx Infrared Imaging System to visualize the protein bands and were analyzed using ImageJ software (National Institutes of Health).

HeLaM cells grown in 12-well dishes were washed in PBS twice, then lysed in 50 µl of RIPA buffer per well containing Protease Inhibitor Cocktail III (Sigma-Aldrich, Poole, UK) and incubated on ice for 10 min. Cell lysates were centrifuged at 4°C and 14,000 g for 10 min to remove debris. Where appropriate, protein concentrations were measured using the bicinchoninic acid reagent (Thermo Fisher Scientific, Cramlington, UK), and equal amounts were loaded onto SDS-PAGE gels. Samples were transferred to PVDF membrane and blotted using IRDye 700CW- and 800C-labelled secondary antibodies from LI-COR Biosciences (Cambridge, UK).