rabbit polyclonal anti hsc70 hspa8  (Proteintech)

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

Journal: Cell reports

Article Title: Chronic hypoxia stabilizes 3βHSD1 via autophagy suppression

doi: 10.1016/j.celrep.2023.113575

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-HSPA8 , Proteintech Group , 10654-1-AP;RRID:AB_2120153.

Techniques: Recombinant, Transfection, Mutagenesis, Viability Assay, Sequencing, Negative Control, shRNA, Plasmid Preparation, Expressing, Variant Assay, Software