Quantity one software 4

(1) The 170 kDa band was excised from the blot and processed for internal sequence analysis as described [13 (link)]. Briefly, in situ digestion was done using 0.01 μg/μL trypsin (in 100 μL of 25 mM NH₄HCO₃) for 30 min at 4 °C, until the trypsin was completely absorbed by the gel particles. Then, the resulting mixture was added to 100 μL of 25 mM NH₄HCO₃, the supernatant was collected by centrifugation and transfered in another Eppendorf tube. The extraction buffer (5% TFA, 95% ddH₂O) was added into the pellets for 1 h, and then treated with an extraction buffer (2.5% TFA, 50% ACN, 47.5% ddH₂O) for 1 h and stored at −20 °C. (2) Mass Spectrometry analysis was carried out using the Tanon-6100 Chemiluminescent Imaging system (Tanon Science and technology Co., Ltd., Shanghai, China). The band densities were calculated with Quantity One software 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The excised gel was digested with trypsin and subjected to LC-MS/MS. The data were recorded on an LTQ-Orbitrap Fusion mass spectrometer (ThermoFisher Scientific Inc., Waltham, MA, USA) coupled to an Easy-nLC 1000 LC System (ThermoFisher Scientific Inc., Waltham, MA, USA). Label-free MS analysis was performed by Thermo Q-Exactive mass spectrometry, and the MS raw data were processed using MaxQuant software with the Uniprot-Orytolagus cuniculus database.

Cells were lysed with a 1% NP-40 hypotonic lysis buffer containing 1 mM phenylmethanesulfonyl fluoride, 1% aprotinin, and 1 mM sodium vanadate (Sigma-Aldrich). Cell lysates were mixed with loading buffer, boiled for 5 min, resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). This membrane was subsequently blocked with 5% nonfat milk. Western blot analysis was then performed in accordance with standard protocols using antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) against 1α-hydroxylase, VDR, LL-37, or β-defensin-2 (HBD2), followed by incubation with relevant horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Relative changes in protein expression were estimated from the mean pixel density using Quantity One software 4.6.2 (Bio-Rad, Hercules, CA, USA), normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and presented as relative density units.

Aortic tissues and EA.hy926 cells were retrieved and subjected to disruption by an ultrasonic homogenizer in RIPA containing protease and phosphatase inhibitors (Pierce Biotechnology, Rockford, IL, USA). Protein concentrations were determined using an assay from Pierce (Rockford, IL, USA). Equal amounts of protein were separated on 10% SDS-PAGE gels; Western blotting was performed as previously described [11 (link)]. The primary antibodies included VCAM-1(1:800), MCP-1 (1:800), Pin1 (1:1000), ERK1/2 (1:1000), p-ERK1/2 (1:1000), JNK (1:1000), p-JNK (1:1000), p65 (1:1000), p-p65 (Ser536; 1:1000), and β-actin (1:5000). Signals were visualized using the enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockford, IL, USA). Quantitative analysis of the band density was performed using Quantity One software 4.6.2 (Bio-Rad, Berkeley, CA, USA). All bands were normalized to β-actin.

Tissue lysates and cell lysates were prepared using RIPA buffer (Solarbio, China) to obtain total proteins. Proteins (50 µg) were separated on SDS-PAGE and transferred on polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% (w/v) skimmed milk solution for 1 h, and incubated with rabbit primary antibodies (Abcam) against sFlt-1, PlGF, SIRT1, eNOS and GAPDH at 4°C for 12 h. Next, the membranes were washed and incubated with appropriate anti-rabbit HRP-conjugated secondary antibodies at 37°C for 1 h. Protein bands were detected using chemiluminescence and band intensities were analyzed using Quantity One software 4.6.2 (Bio-Rad, Hercules, CA, USA).