solute carrier family 6 member 8  (Proteintech)

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) Volcano plot of differentially expressed genes from RNA sequencing, showing upregulation of PCSK9 and downregulation of APOA4 in the LD+TMAO group compared to the LD group. (B) qRT-qPCR analysis of APOA4 mRNA expression levels across groups (n = 3 per group). (C) qRT-qPCR analysis of PCSK9 mRNA levels across groups (n = 3 per group). (D) Western blot analysis of APOA4 and PCSK9 protein levels across groups (n = 3 per group). (E–F). Quantification results of the Western blot analysis using ImageJ. (G, H). Serum APOA4 and PCSK9 levels in patients with cholelithiasis and the control group detected by ELISA. ** p < 0.01, *** p < 0.001. LD, lithogenic diet; TMAO, trimethylamine-N-oxide; NC, negative control; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; GSD, gallstone disease; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) Lithogenesis in mice from the LD and LD+DMB groups. (B) Weights of solid contents in the gallbladders of mice from each group. (C) Serum TMAO levels in the LD and LD+DMB groups detected by ELISA. (D) Immunofluorescence staining of APOA4 and PCSK9 in hepatic tissues from the LD and LD+DMB groups. (E, F) qRT-qPCR analysis of APOA4 and PCSK9 mRNA expression levels in hepatic tissues from the LD and LD+DMB groups. * p < 0.05, ** p < 0.01, **** p < 0.0001. LD, lithogenic diet; DMB, 3,3-dimethyl-1-butanol; TMAO, trimethylamine-N-oxide; ELISA, enzyme-linked immunosorbent assay; APOA4, apolipoprotein A4; PCSK9 proprotein convertase subtilisin/kexin type 9.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A–E) APOA4 , PCSK9 , HMGCR, ABCG5 , and ABCG8 mRNA expression levels in TMAO-treated and control AML12 cells (n = 3 per group). (F) Immunofluorescence staining for APOA4, PCSK9, HMGCR, ABCG5, and ABCG8 in cells from each group. ** p < 0.01, *** p < 0.001. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Expressing, Control, Immunofluorescence, Staining, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) WB analysis of PCSK9, HMGCR, ABCG5, and ABCG8 in NC, TMAO-treated, and APOA4-overexpressed cells. (B–E) Changes in PCSK9 , HMGCR , ABCG5 , and ABCG8 mRNA levels after APOA4 overexpression (n = 3 per group). (F) Immunofluorescence staining for intracellular PCSK9, HMGCR, ABCG5, and ABCG8 after APOA4 overexpression. (G) WB analysis of APOA4, HMGCR, ABCG5, and ABCG8 in NC, TMAO-treated, and PCSK9-knockdown cells. (H–K) Changes in mRNA levels of PCSK9 , HMGCR , ABCG5 , and ABCG8 after PCSK9 knockdown (n = 3 per group). (L) Immunofluorescence staining for PCSK9, HMGCR, ABCG5, and ABCG8 in cells after PCSK9 knockdown. * p < 0.05, ** p < 0.01. +, positive expression; -, negative expression; TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Over Expression, Immunofluorescence, Staining, Knockdown, Expressing, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: Gut microbiota produce trimethylamine, which enters the liver via the portal circulation and is primarily oxidized by FMO3 to produce TMAO. TMAO upregulates hepatic PCSK9 gene expression while downregulating APOA4 expression. PCSK9 overexpression inhibits APOA4 expression, while low APOA4 expression further promotes PCSK9 expression, forming a feedback loop that dysregulates cholesterol metabolism. This upregulates cholesterol synthesis by HMGCR and cholesterol efflux by ABCG5/8. Consequently, biliary concentrations of cholesterol and bile acids increase and decrease, respectively, thereby promoting cholesterol gallstone formation. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; FMO3, flavin containing monooxygenase 3; TMA, Trimethylamine.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Gene Expression, Expressing, Over Expression, Binding Assay

Journal: Veterinary Research

Article Title: Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation

doi: 10.1186/s13567-024-01392-w

Figure Lengend Snippet: The primers used for RT-qPCR in this study

Article Snippet: Rabbit anti-heat shock protein member 8 (HSPA8) polyclonal antibodies (pAbs; Cat. No. 10654–1-AP), mouse anti-lysosomal-associated membrane protein 2A (LAMP2A) mAb (Cat. No. 66301–1-Ig), rabbit anti-β-actin mAb (Cat. No. 81115–1-RR), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Cat. No. 60004–1-Ig) were purchased from Proteintech.

Techniques: Sequencing

Journal: Veterinary Research

Article Title: Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation

doi: 10.1186/s13567-024-01392-w

Figure Lengend Snippet: PRRSV Nsp2 interacts with HSPA8 . A HEK-293 T cells were transfected with the plasmid expressing HA or Nsp2-HA. The proteins were immunoprecipitated in cell lysates using an anti-HA antibody, separated by 12% SDS-PAGE, and stained with silver. The red arrow indicates the significantly different immunoprecipitated protein band. The black arrow marks Nsp2-HA. The panel on the right shows the tandem MS analysis of HSPA8 peptides. B HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and HSPA8-myc or HA/myc-tagged empty vector for 36 h, followed by co-IP with anti-HA or anti-myc magnetic beads, and IB analyses with anti-HA and anti-myc antibodies. C HeLa cells were transfected with the plasmids encoding Nsp2-HA and HSPA8-myc for 24 h. In parallel, HeLa cells were transfected with the plasmid encoding Nsp2-HA and myc-tagged empty vector, or HSPA8-myc and HA-tagged empty vector. HSPA8-myc and Nsp2-HA were visualised with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. The fluorescent signals were observed with confocal microscopy (scale bars = 10 µm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. D MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. They were then analysed via endogenous IP using protein A/G magnetic beads pre-incubated with anti-Nsp2 pAbs, and IB with anti-Nsp2 and anti-HSPA8 antibodies. E MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. The fluorescent signals were observed with confocal microscopy (scale bars = 10 µm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software.

Article Snippet: Rabbit anti-heat shock protein member 8 (HSPA8) polyclonal antibodies (pAbs; Cat. No. 10654–1-AP), mouse anti-lysosomal-associated membrane protein 2A (LAMP2A) mAb (Cat. No. 66301–1-Ig), rabbit anti-β-actin mAb (Cat. No. 81115–1-RR), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Cat. No. 60004–1-Ig) were purchased from Proteintech.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Staining, Co-Immunoprecipitation Assay, Magnetic Beads, Confocal Microscopy, Software, Infection, Incubation

Journal: Veterinary Research

Article Title: Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation

doi: 10.1186/s13567-024-01392-w

Figure Lengend Snippet: PRRSV Nsp2 degrades TBK1 via CMA . A HEK-293 T cells were transfected with the plasmids encoding Flag-TBK1, HSPA8-myc, and Nsp2-HA or HA-tagged empty vector for 36 h. Co-IP was performed with anti-myc magnetic beads and IB was conducted with the specific antibodies. B HeLa cells were transfected with the plasmids encoding Flag-TBK1, HSPA8-myc, and Nsp2-HA or HA-tagged empty vector for 36 h. The fluorescent signals were observed with confocal microscopy. The co-localisation was assessed by determination of the Pearson’s correlation coefficient (scale bars = 10 µm) using the JaCoP plugin in ImageJ software. C MARC-145 cells were infected with PRRSV at 0.1 MOI for 24 h. The fluorescent signals were observed with confocal microscopy. The co-localisation was assessed by determination of the Manders’ correlation coefficient (scale bars = 10 µm) using the JaCoP plugin in ImageJ software. D HEK-293 T cells were transfected with siHSPA8, siLAMP2A, or siNC, and the plasmids encoding Flag-TBK1 and Nsp2-HA. The samples were collected after 36 h for IB analyses with the specific antibodies. E MARC-145 cells were transfected with siHSPA8/LAMP2A or siNC to detect their effects during PRRSV infection, and IB was conducted with the specific antibodies. The data are presented as means ± SEM from three independent experiments. Statistical analysis was carried out using the Student t test. **, P < 0.01, ****, P < 0.0001.

Article Snippet: Rabbit anti-heat shock protein member 8 (HSPA8) polyclonal antibodies (pAbs; Cat. No. 10654–1-AP), mouse anti-lysosomal-associated membrane protein 2A (LAMP2A) mAb (Cat. No. 66301–1-Ig), rabbit anti-β-actin mAb (Cat. No. 81115–1-RR), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Cat. No. 60004–1-Ig) were purchased from Proteintech.

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Magnetic Beads, Confocal Microscopy, Software, Infection

Journal: Veterinary Research

Article Title: Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation

doi: 10.1186/s13567-024-01392-w

Figure Lengend Snippet: Schematic model depicting that PRRSV inhibits IFN-I production by degrading TBK1 via CMA, thereby promoting viral proliferation . Mechanistically, PRRSV Nsp2 enhances the interaction between HSPA8 and TBK1, leading to LAMP2A-mediated translocation of TBK1 into lysosomes for degradation, which impedes downstream IRF3 signalling and IFN-I production.

Article Snippet: Rabbit anti-heat shock protein member 8 (HSPA8) polyclonal antibodies (pAbs; Cat. No. 10654–1-AP), mouse anti-lysosomal-associated membrane protein 2A (LAMP2A) mAb (Cat. No. 66301–1-Ig), rabbit anti-β-actin mAb (Cat. No. 81115–1-RR), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Cat. No. 60004–1-Ig) were purchased from Proteintech.

Techniques: Translocation Assay