Cells were lysed in RIPA lysis buffer containing protease inhibitors (Thermo Fisher Scientific). Cell protein lysates were centrifuged at 14,000 rpm for 20 min at 4°C and the supernatants were collected. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein from samples were resolved on 4–20% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE; Applygen Technologies) and transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking for nonspecific binding with 5% non-fat dry milk for 1 h at room temperature, the membranes were incubated with antibody HNF1α (1:500, ab96777, Abcam, Cambridge, MA, USA), SCAP (1:2000, ab125186, Abcam), SREBP-2 (1:500, ab30682, Abcam), PCSK9 (1:1000, ab181142, Abcam), or β-actin (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C and followed by an incubation with secondary antibody (1:3000, #7074, Cell Signaling Technology) for 1 h at room temperature. Protein was visualized with enhanced chemiluminescence (ECL; Thermo Fisher Scientific) and membranes were imaged with GE Healthcare imaging system (Waukesha, WI, USA). Band intensities were quantified using Image J software.
Imaging system
Manufactured by GE Healthcare
Sourced in United States
The Imaging system is a medical imaging device designed to capture and generate visual representations of the internal structures of the human body. It utilizes various imaging technologies to produce high-quality images for diagnostic and clinical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab96777, by Abcam (1 mentions)
Ab30682, by Abcam (1 mentions)
Ab181142, by Abcam (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti sox9, by Abcam (1 mentions)
Anti gsk3β, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Premix, by Bioneer (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Pcr premix, by Bioneer (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Anti rabbit secondary antibody, by Bio-Rad (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
8 protocols using imaging system
1
Western Blot Analysis of Cholesterol Regulators
2
MRI Brain Morphometry Analysis Pipeline
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Participants were scanned using a 3.0 T General Electric imaging system (Milwaukee, United States). A three-dimensional T1-weighted fast spoiled gradient echo (FSPGR) sequence was acquired in sagittal plane (repetition time/echo time/inversion time = 8.73/1.74/650 ms, field of view = 250 × 250 mm, matrix = 250 × 250 mm, flip angle = 12°, slice thickness = 1 mm).
The T1-weighted images were processed with FreeSurfer 5.1.0 (http://surfer.nmr.mgh.harvard.edu/ ) through our database system (theHiveDB)25 (link) as detailed elsewhere26 (link). Careful visual quality control was performed on both the original and the processed data, and manual edits were done when appropriate to ensure optimal output. Measures of thickness were calculated for 34 cortical regions from both hemispheres27 (link), and measures of volume for 21 subcortical regions28 (link). A measurement of total intracranial volume (ICV) was also estimated with FreeSurfer in order to account for individual differences in brain size on all the volumetric measures29 (link).
The T1-weighted images were processed with FreeSurfer 5.1.0 (
3
Targeted Proteomics and Western Blot Analysis
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For PRM analysis, protein treatment was performed in the same way as in the iTRAQ experiment. The peptide information was imported into the software Xcalibur for PRM method setting. Chromatographic separation was carried out using high performance liquid chromatography (HPLC) system. The samples separated by HPLC were subjected to PRM mass spectrometry using a Q-Active HF mass spectrometer (Thermo Scientific). Finally, Skyline 3.5.0 software was used to analyze the original PRM files.
For Western blot assay, the procedure of cell lysate, protein quantification, separation and transfer were performed as before (Li et al., 2018 (link)). Then, membranes were blocked in skim milk at room temperature (RT) and probed overnight with primary antibodies including anti-YAP1 (1:2000; cat. no. ab109307; Abcam), anti-SOX9 (1:2000; cat. no. ab185966; Abcam), anti-PRKD3 (1:2000; cat. no. ab252982; Abcam), anti-GSK-3β (1:2000; cat. no. ab32391; Abcam), and anti-β-actin (1:1,000; cat. no. TA-09; Zhongshan Jinqiao Bio-Technology Co., Ltd.) as the internal reference. After incubation with secondary antibody at RT for 1.5 h, the PVDF membranes were incubated with ECL reagent. The images were captured in imaging system (GE Healthcare Life Sciences).
For Western blot assay, the procedure of cell lysate, protein quantification, separation and transfer were performed as before (Li et al., 2018 (link)). Then, membranes were blocked in skim milk at room temperature (RT) and probed overnight with primary antibodies including anti-YAP1 (1:2000; cat. no. ab109307; Abcam), anti-SOX9 (1:2000; cat. no. ab185966; Abcam), anti-PRKD3 (1:2000; cat. no. ab252982; Abcam), anti-GSK-3β (1:2000; cat. no. ab32391; Abcam), and anti-β-actin (1:1,000; cat. no. TA-09; Zhongshan Jinqiao Bio-Technology Co., Ltd.) as the internal reference. After incubation with secondary antibody at RT for 1.5 h, the PVDF membranes were incubated with ECL reagent. The images were captured in imaging system (GE Healthcare Life Sciences).
4
ApoE-KO Mice Plaque Imaging by Au DENPs
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Animal experiments and animal care were in accordance with the protocols of Shanghai JiaoTong University School of Medicine. ApoE-KO (C57BL/6 background) mice and C57 mice were purchased from Shanghai Research Center for Model Organisms (Shanghai, People’s Republic of China). Male ApoE-KO mice (n=9) with a mean age of 11±1.6 months were fed a Western diet (containing 21% fat, 0.15% cholesterol) for at least 6 months before the experiment. The mice were anesthetized using an intraperitoneal dose of pentobarbital sodium (40 mg/kg). Au DENPs (0.4 μmol Au per g body weight, [Au] =0.2 M) dispersed in PBS were then given intravenously into the mice tail veins. Age-matched C57 mice were used as the negative control (n=3). All mice underwent a baseline scan at first, followed by micro-CT scans at 20 minutes and 2, 4, and 6 hours postinjection using a GE Healthcare imaging system.
5
Quantifying Hepatic Cholesterol Metabolism Genes
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For the mRNA expression of SREBP-2, ACAT-2, and HMG-CoA, total RNA was extracted from liver tissue with TRIZOL solution using a homogenizer as previously described [19 (link)]. The RNA (2 μg) was annealed with OligodT by heating at 70°C for 10 min and kept in ice for cooling for another 10 min. The reverse transcriptase reaction was carried out using a commercially available premix (Bioneer, Daejeon, Republic of Korea) by heating at 42°C for 1.5 h, and the reaction was stopped by heating at 95°C for 5 min. The resulting cDNA was added to the PCR premix (Bioneer, Daejeon, Republic of Korea) with the respective target gene primers. The PCR product was run on 1% agarose gel stained with ethidium bromide (EtBr), and the gel images were developed using an imaging system (GE Healthcare, Chicago, IL, USA). The band intensities were normalized against GAPDH, which was used as the housekeeping gene. Similarly, after reverse transcriptase, the resultant cDNA was then added with SYBR Green (Thermo Fisher, Waltham, MA, USA) with primers of GAPDH, ACAT-1, and ACAT-2 for real-time PCR analysis using CFX96 Real Time System (Biorad, Hercules, CA, USA) as previously described [19 (link)].
6
Western Blot Analysis of Cell Signaling
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Western blot analysis was performed following the previously established protocol (Hu et al., 2023) . Briefly, the proteins were separated on a sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel before being deposited onto a transfer membrane (Pall Corporation, Pensacola, FL, USA). The membranes were then blocked for a duration of 45 min in the presence of 5% nonfat milk at room temperature. Primary antibodies against phospho-STAT-3 (Tyr705) (p-STAT3), phospho-extracellular signal-regulated kinase1/2 (ERK1/2) (Thr202/Tyr204) (p-ERK1/2), phospho-glycogen synthase kinase-3β (Ser9) (p-GSK-3β), phospho-AKT (Ser473) (p-AKT), STAT-3, ERK1/2, GSK-3β, and AKT (all 1:1000, Cell Signaling Technology, Danvers, MA, USA) were used in the study. The membranes were subsequently subjected to a two-hour incubation with a secondary anti-rabbit antibody (1:5000, Bio-Rad, Hercules, CA, USA). The bands were detected by a chemiluminescent ECL reagent (Beijing 4A Biotech Co., Ltd., Beijing, China). An imaging system (GE Healthcare, Little Chalfont, UK) was used to detect the signals.
The phosphorylation signal densities were normalized by comparing the band densities to the overall protein signal densities via ImageJ (National Institutes of Health, Bethesda, MD, USA).
The phosphorylation signal densities were normalized by comparing the band densities to the overall protein signal densities via ImageJ (National Institutes of Health, Bethesda, MD, USA).
7
Quantification of Protein Levels by Western Blot
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Western blot was performed based on a previously described method [22 (link)]. In short, RAW 264.7 cells were inoculated into a 6-well plate (1 × 105 cells/mL) and treated with LPS (1 µg/mL) and PFF (0, 50, 100, and 200 μg/mL) for 24 h. After incubation, the cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer. After centrifugation (12,000× g, 4 °C, 20 min), the protein concentration of the supernatant was quantified by BCA Kit. Meanwhile, the samples and ×5 loading buffer were mixed and heated (100 °C, 10 min) to denature the protein completely. Then, 50 μg of total protein was separated by SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane, which was blocked by 5% BSA and incubated with the primary antibodies. After that, membranes were further incubated with the secondary antibody. Finally, protein bands were reacted with ECL solutions and visualized using an imaging system (GE, Chicago, IL, USA).
8
Western Blot Analysis of ISOC1 Protein
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Transfected cells were lysed and total proteins were extracted using cell lysis buffer for Western blot and immunoprecipitation (Beyotime Biotechnology, China). Equal amounts of proteins were separated by 10–12% SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with tris-buffered saline (TBS) solutions (containing 5% fat-free milk powder and 10% Tween 20) at room temperature for 1 h and incubated with ISOC1 (1:1,000 dilution; Abcam, USA) or beta-actin (1:2,000 dilution; Abcam, USA) overnight at 4 °C, followed with secondary antibody (1:5,000 dilution; Cell Signaling Technology, USA) at room temperature for 2 h. The blots were visualized using enhanced chemiluminescence (ECL) solutions and an imaging system (GE Healthcare, USA).
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