Journal: Nutrients

Article Title: Integrated Proteomics and Metabolomics Reveal the Direct Hepatic Protection of Propionate Against Alcoholic Liver Disease via the RGN-PPARα Pathway

doi: 10.3390/nu18050872

Figure Lengend Snippet: The effect of PA on lipid accumulation in ALD mice. ( A ) Schematic of the acute alcoholic liver injury model in mice with propionate treatment. ( B , C ) Serum ALT and AST levels in mice. ( D , E ) H&E and Oil Red O staining of liver tissues (scale bar: 50 μm). ( F ) The relative area of Oil Red O staining. ( G , H ) Liver levels of TC and TG in mice. ( I – L ) Serum concentrations of TC, TG, HDL-C and LDL-C in mice. ( M ) Representative Western blot analysis and ( N ) quantification of ACC1, SREBP1 and CPT1A protein expression in liver tissues normalized to β-actin (n = 4). The data are presented as means ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies, including β-actin (#66009-1-Ig), CPT1A (#15184-1-AP), SREBP-1 (#14088-1-AP), ACC1 (#21923-1-AP), ACOX1 (#83731-2-RR), PPARα (#66826-1-Ig) and RGN (#17947-1-AP), were obtained from Proteintech Group (Wuhan, China).

Techniques: Staining, Western Blot, Expressing

Journal: Nutrients

Article Title: Integrated Proteomics and Metabolomics Reveal the Direct Hepatic Protection of Propionate Against Alcoholic Liver Disease via the RGN-PPARα Pathway

doi: 10.3390/nu18050872

Figure Lengend Snippet: Propionate suppressed cell injury and lipid accumulation in vitro. ( A ) Changes in AML-12 cell viability after a series of doses of ethanol challenge. ( B ) Changes in AML-12 cell viability after a series of concentrations of OA incubation. ( C ) Effects of different concentrations of propionate on the viability of AML-12 cells. ( D ) Propionate restored AML-12 cells’ viability following co-treatment with 200 mM ethanol and 0.5 mmol/L OA. ( E ) Representative views of Oil Red O staining of AML-12 cells (scale bars: 100 μm and 50 μm). ( F ) Representative images of Western blot analysis and ( G ) quantification of ACC1, SREBP1 and CPT1A protein expression in AML-12 cells normalized to β-actin (n = 3). The data are presented as means ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies, including β-actin (#66009-1-Ig), CPT1A (#15184-1-AP), SREBP-1 (#14088-1-AP), ACC1 (#21923-1-AP), ACOX1 (#83731-2-RR), PPARα (#66826-1-Ig) and RGN (#17947-1-AP), were obtained from Proteintech Group (Wuhan, China).

Techniques: In Vitro, Incubation, Staining, Western Blot, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PI3K p85α/HIF-1α accelerates the development of pulmonary arterial hypertension by regulating fatty acid uptake and mitophagy.

doi: 10.1186/s10020-024-00975-9

Figure Lengend Snippet: Fig. 1 HIF-1α was closely associated with the Parkin/PINK1 axis and fatty acid metabolism-related factors in PAH patients. A Serum levels of HIF-1α were measured by ELISA. B Serum levels of Parkin and PINK1 were measured by ELISA. C Serum levels of FAS, ACC, and CD36 were measured by ELISA. *P < 0.05 vs. Normal. The statistical test was performed using unpaired Student’s t-test. D Spearman’s correlation analysis was performed to determine the correlation between HIF-1α, Parkin, PINK1, FAS, ACC, and CD36. *P < 0.05. ***P < 0.001. ****P < 0.0001

Article Snippet: The levels of HIF-1α, Parkin, PINK1, fatty acid synthetase (FAS), acetyl-CoA carboxylase (ACC), and CD36 in human or rat serum were measured according to the instructions of the following ELISA kits: Human HIF-1α ELISA kit (CSB-E12112h, CUSABIO Co., Ltd., Wuhan, China), Human Parkin ELISA Kit (JL11195, Shanghai Jianglai Industrial Limited by Share Ltd., Shanghai, China), Human PINK1 ELISA Kit (JL11175, Shanghai Jianglai Industrial Limited by Share Ltd.), Human/Rat FAS ELISA Kit (CSB-E04542h/CSB-E07324r, CUSABIO Co., Ltd.), Human/Rat ACC ELISA Kit (CSBEL001119HU, CUSABIO Co., Ltd./YJ059228, Shanghai Yuanju Biotechnology Center, Shanghai, China), and Human/Rat CD36 ELISA Kit (CSB-E10117h/CSBEL004927RA, CUSABIO Co., Ltd.).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of international medical research

Article Title: The therapeutic potential of irisin in alleviating acute lung injury via inflammation and ferroptosis modulation.

doi: 10.1177/03000605251340338

Figure Lengend Snippet: Figure 6. Western blotting analysis on the effects of irisin on the expression level of ferroptosis-associated proteins. (a) Representative western blotting images for the expression of ACSL4, COX-2, GPX4, p-AMPK, and t-AMPK in lung tissues. GAPDH was selected as the loading control protein. (b) Quantification analysis of the related bands of ACSL4, COX-2, GPX4, p-AMPK, and t-AMPK in lung tissues. Lung tissues were harvested on day 3 post-CLP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. n ¼ 6 per group. Data are presented as means SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Subsequently, the membranes were incubated with rabbit anti-AMPK (MCE, Cat# HY-P80541), anti-p-AMPK (MCE, HYP80452), GPX4 (MCE, HY-P80450), ACSL4 (Santa Cruz Biotechnology, Dallas, Texas, USA; Cat# sc-365230), COX-2 (Abcam, Cambridge, United Kingdom; Cat# ab283574), and GAPDH (MCE, HY-P80137) antibodies at 4 C overnight.

Techniques: Western Blot, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: Comparative proteomic and genomic analyses of Brucella abortus biofilm and planktonic cells

doi: 10.3892/mmr.2019.10888

Figure Lengend Snippet: Differentially expressed proteins in the Brucella abortus A3313 biofilm, identified by MALDI-TOF/TOF-MS analysis.

Article Snippet: Then, the blocked membrane was incubated with sera from primary antibodies [hypothetical protein BAbS19_I16470 (1:1,000; cat. no. orb309412; Biorbyt Ltd.); chaperone protein DnaJ (1:1,000; cat. no. PA3-018; Invitrogen; Thermo Fisher Scientific, Inc.); elongation factor Tu (1:1,000; cat. no. ab210089; Abcam); Chaperonin Cpn60/TCP-1 (1:1,000; cat. no. 3094R-100; BioVision, Inc.); polyprenyl synthetase (1:1,000; cat. no. ab80647; Abcam); periplasmic binding protein (1:1,000; cat. no. M30934-1; Wuhan Boster Biological Technology, Ltd.); enolase (1:1,000; cat. no. sc-271384; Santa Cruz Biotechnology, Inc.); acetyl-CoA carboxylase, a subunit (1:1,000; cat. no. MAB6898; R&D Systems, Inc.); tryptophanyl-tRNA synthetase (1:1,000; cat. no. ab31536; Abcam); aspartate-semialdehyde dehydrogenase (1:1,000; cat. no. EM1708-10a; Jingke Huaxue; exosporium protein B (1:1,000; cat. no. ab92932; Abcam); enoyl-(acyl carrier protein) reductase (1:1,000; cat. no. abx109426; Abbexa Ltd.); Omp16 (1:1,000; cat. no. ab93127; Abcam)] for 2 h at room temperature and then incubated with horseradish peroxidase-labeled secondary antibodies (1:5,000; cat. no. 29139; Invitrogen; Thermo Fisher Scientific, Inc.) in blocking buffer for 1 h at room temperature.

Techniques: Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Comparative proteomic and genomic analyses of Brucella abortus biofilm and planktonic cells

doi: 10.3892/mmr.2019.10888

Figure Lengend Snippet: Differential expression of mRNAs between Brucella abortus cultured under planktonic or biofilm conditions. The relative expression levels of (A) 18 genes encoding proteins identified by 2-D electrophoresis and (B) 14 genes identified via high-throughput sequencing. 2-D, two-dimensional. *P<0.05 vs. control (planktonic cells). E11-22, hypothetical protein BAbS19_I16470; E15-1, polyprenyl synthetase; E21, ExsB protein; J12, Chaperonin Cpn60TCP-1; E13-1, elongation factor Tu; E20-2, aspartate-semialdehyde dehydrogenase; C24-1, enoyl-(acyl carrier protein) reductase; E12-1, DnaJ, chaperone protein DnaJ; E12-3, isocitrate dehydrogenase; E17, Enolase; E18, Acetyl-CoA carboxylase, alpha subunit; C24-2, putative sulfite oxidase subunit YedY; E11-1, Bacterial protein export chaperone SecB; E13-2, Antifreeze protein, type I; E15-2, Lactatemalate dehydrogenase; E15-5, Phosphoribosylformylglycinamidinecyclo-ligase; E16-3, Periplasmic binding protein; E19-2, tryptophanyl-tRNAsynthetase.

Article Snippet: Then, the blocked membrane was incubated with sera from primary antibodies [hypothetical protein BAbS19_I16470 (1:1,000; cat. no. orb309412; Biorbyt Ltd.); chaperone protein DnaJ (1:1,000; cat. no. PA3-018; Invitrogen; Thermo Fisher Scientific, Inc.); elongation factor Tu (1:1,000; cat. no. ab210089; Abcam); Chaperonin Cpn60/TCP-1 (1:1,000; cat. no. 3094R-100; BioVision, Inc.); polyprenyl synthetase (1:1,000; cat. no. ab80647; Abcam); periplasmic binding protein (1:1,000; cat. no. M30934-1; Wuhan Boster Biological Technology, Ltd.); enolase (1:1,000; cat. no. sc-271384; Santa Cruz Biotechnology, Inc.); acetyl-CoA carboxylase, a subunit (1:1,000; cat. no. MAB6898; R&D Systems, Inc.); tryptophanyl-tRNA synthetase (1:1,000; cat. no. ab31536; Abcam); aspartate-semialdehyde dehydrogenase (1:1,000; cat. no. EM1708-10a; Jingke Huaxue; exosporium protein B (1:1,000; cat. no. ab92932; Abcam); enoyl-(acyl carrier protein) reductase (1:1,000; cat. no. abx109426; Abbexa Ltd.); Omp16 (1:1,000; cat. no. ab93127; Abcam)] for 2 h at room temperature and then incubated with horseradish peroxidase-labeled secondary antibodies (1:5,000; cat. no. 29139; Invitrogen; Thermo Fisher Scientific, Inc.) in blocking buffer for 1 h at room temperature.

Techniques: Quantitative Proteomics, Cell Culture, Expressing, Electrophoresis, Next-Generation Sequencing, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Comparative proteomic and genomic analyses of Brucella abortus biofilm and planktonic cells

doi: 10.3892/mmr.2019.10888

Figure Lengend Snippet: RT-qPCR identification of the mRNA expression levels of 18 proteins identified in 2-D electrophoresis.

Article Snippet: Then, the blocked membrane was incubated with sera from primary antibodies [hypothetical protein BAbS19_I16470 (1:1,000; cat. no. orb309412; Biorbyt Ltd.); chaperone protein DnaJ (1:1,000; cat. no. PA3-018; Invitrogen; Thermo Fisher Scientific, Inc.); elongation factor Tu (1:1,000; cat. no. ab210089; Abcam); Chaperonin Cpn60/TCP-1 (1:1,000; cat. no. 3094R-100; BioVision, Inc.); polyprenyl synthetase (1:1,000; cat. no. ab80647; Abcam); periplasmic binding protein (1:1,000; cat. no. M30934-1; Wuhan Boster Biological Technology, Ltd.); enolase (1:1,000; cat. no. sc-271384; Santa Cruz Biotechnology, Inc.); acetyl-CoA carboxylase, a subunit (1:1,000; cat. no. MAB6898; R&D Systems, Inc.); tryptophanyl-tRNA synthetase (1:1,000; cat. no. ab31536; Abcam); aspartate-semialdehyde dehydrogenase (1:1,000; cat. no. EM1708-10a; Jingke Huaxue; exosporium protein B (1:1,000; cat. no. ab92932; Abcam); enoyl-(acyl carrier protein) reductase (1:1,000; cat. no. abx109426; Abbexa Ltd.); Omp16 (1:1,000; cat. no. ab93127; Abcam)] for 2 h at room temperature and then incubated with horseradish peroxidase-labeled secondary antibodies (1:5,000; cat. no. 29139; Invitrogen; Thermo Fisher Scientific, Inc.) in blocking buffer for 1 h at room temperature.

Techniques: Expressing, Electrophoresis, Binding Assay

Journal: iScience

Article Title: Characterization of an in vitro steatosis model simulating activated de novo lipogenesis in MAFLD patients

doi: 10.1016/j.isci.2023.107727

Figure Lengend Snippet: The relevance of the DNL steatosis model to the development of MAFLD in humans There are exogenous (blue box) and endogenous (red box) origins of fatty acid accumulation in the liver. For de novo lipogenesis, dietary glucose is metabolized to pyruvate and enters mitochondria for energy production. Then citrate is transported into the cytoplasm, where the citrate is catalyzed to Acetyl-CoA by the ATP citrate lyase (ACLY). Then, ACACA converts acetyl-CoA to Malonyl-CoA. Subsequently, fatty acid synthase (FASN) adds malonyl-CoA to the growing fatty acid chain to form palmitate. Stearoyl CoA desaturase (SCD), Fatty Acid Desaturase 1 (FADS1), and Fatty Acid Desaturase 1 (FADS2) further convert palmitate to long-chain fatty acids. Glycerol-3-Phosphate Acyltransferase, Mitochondrial (GPAM) further converts fatty acid to TAGs, which may be stored in the liver. Hyperinsulinemia induces the SREBP-1C expression and hyperglycemia activates the ChREBP-1C expression, leading to the transcriptional activation of all the lipogenic genes. On the other hand, a free fatty acid from dietary lipids or adipocyte lipolysis could be directly assembled into TAGs or enter mitochondria for energy production and be transported to the cytoplasm for flowing de novo lipogenesis. Our results showed that key genes and transcription factors associated with the glycolysis DNL were significantly upregulated in the DNL steatosis model compared with the OA steatosis model (marked in red), indicating that the DNL steatosis model could better reflect the molecular features of MAFLD, and it might be better in vitro model for MAFLD studies.

Article Snippet: FASN (ab22759, abcam), ACACA (NBP2-55439, Novus), ChREBP (ab92809, abcam), SREBP-1C (PA1 337, Invitrogen), PKL (06653, Sigma), PKM (4053S, Cell signalling), Tyr-105 p-PKM (3827S, Cell signaling), β-actin (ab8227, abcam), α-tubulin (ab7291, abcam) were blotted as a primary antibody for overnight.

Techniques: Expressing, Activation Assay, In Vitro

Journal: iScience

Article Title: Characterization of an in vitro steatosis model simulating activated de novo lipogenesis in MAFLD patients

doi: 10.1016/j.isci.2023.107727

Figure Lengend Snippet:

Article Snippet: FASN (ab22759, abcam), ACACA (NBP2-55439, Novus), ChREBP (ab92809, abcam), SREBP-1C (PA1 337, Invitrogen), PKL (06653, Sigma), PKM (4053S, Cell signalling), Tyr-105 p-PKM (3827S, Cell signaling), β-actin (ab8227, abcam), α-tubulin (ab7291, abcam) were blotted as a primary antibody for overnight.

Techniques: Recombinant, Cell Counting, Software