polyclonal antibodies against dnajb11 peptide arirkknegmpvyn  (GenScript corporation)

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A) Structural features and domains of DnaJB11 and BAP31 from S . furcifera . (B) Interactions among P7-1 of SRBSDV, BAP31 and DnaJB11 were detected by yeast two-hybrid assay. Transformants on plate of QDO culture medium were labeled as follows: P7-1+DnaJB11N, pGBKT7-P7-1/pGADT7-DnaJB11N; P7-1+DnaJB11C, pGBKT7-P7-1/pGADT7-DnaJB11C; P7-1+BAP31, pGBKT7-P7-1/pGADT7-BAP31; BAP31+DnaJB11N, pGBKT7-BAP31/pGADT7-DnaJB11N; BAP31+DnaJB11C, pGBKT7-BAP31/pGADT7-DnaJB11C; Positive control, pGBKT7-53/pGADT7-T; Negative control, pGBKT7-Lam/pGADT7-T. Serially diluted yeast cultures were shown. (C) Both DnaJB11C and BAP31 interacted with the TM1-containing N-terminus, but not with the TM2-containing C-terminus of SRBSDV P7-1, as detected by yeast two-hybrid assay. Transformants on plate of DDO or QDO+X-α-Gal culture medium. (D-G) The interactions among P7-1, BAP31 and DnaJB11C were demonstrated by Co-IP assay. (D) The lysate was immunoprecipitated with P7-1 antibodies, and the immunoprecipitated proteins were detected by BAP31 antibodies. (E) The lysate was immunoprecipitated with P7-1 antibodies, and the immunoprecipitated proteins were detected by DnaJB11 antibodies. (F) The lysate was immunoprecipitated with BAP31 antibodies, and the immunoprecipitated proteins were detected by DnaJB11 antibodies. (G) The lysate was immunoprecipitated with P7-1 antibodies, and the immunoprecipitated proteins were detected by GFP antibodies. (H, I) The competitive interactions among P7-1 of SRBSDV, BAP31 and DnaJB11C were detected by pull-down assay. P7-1-His and MBP-BAP31 were incubated with Ni-NTA agarose beads, then GST-DnaJB11C was added to the beads; when increased the amounts of DnaJB11C, the binding between P7-1 and BAP31 was decreased (H). P7-1-His and GST-DnaJB11C were incubated with Ni-NTA agarose beads, then MBP-BAP31 was added to the beads; when increased the amounts of MBP-BAP31, the binding between P7-1 and DnaJB11C was not affected (I).

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Y2H Assay, Labeling, Positive Control, Negative Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Incubation, Binding Assay

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A) Sf9 cells were fixed at 18, 24, 36, or 48 hpi and immunolabeled with P7-1-FITC (green). (B) After staining with the ER-Tracker Dyes (red) for 30 min, Sf9 cells were fixed at 18 or 36 hpi and immunolabeled with P7-1-FITC (green). (C) After the incubation with 500, 250, or 125 ng/mL BFA or DMSO, Sf9 cells were fixed at 36 hpi and immunolabeled with P7-1-FITC (green). (D) After staining with the ER-Tracker Dyes (red) for 30 min, 500 ng/mL BFA-treated Sf9 cells were fixed at 36 hpi and immunolabeled with P7-1-FITC (green). (E) After staining with the ER-Tracker Dyes (red) for 30 min, Sf9 cells expressed with P7-1N or P7-1C were fixed at 36 hpi and immunolabeled with P7-1-FITC (green). (F) At 48 hpi. Sf9 cells expressed with BAP31 or DnaJB11C were fixed and immunolabeled with BAP31-FITC (green) or DnaJB11-FITC (green), respectively. (G) At 48 hpi, Sf9 cells co-expressed with BAP31 and DnaJB11C were stained with the ER-Tracker Dyes (red), and then fixed and immunolabeled with BAP31-FITC (green) and DnaJB11-specifc IgG conjugated to Alexa Fluor 647 (DnaJB11-Alexa Fluor 647, blue). (H) At 18 or 48 hpi, Sf9 cells co-expressed with BAP31 and P7-1 were fixed and immunolabeled with BAP31-FITC (green) and P7-1-rhodamine (red). (I) At 18 or 48 hpi, Sf9 cells co-expressed with P7-1 and DnaJB11C or the complete DnaJB11 were fixed and immunolabeled with P7-1-FITC (green) and DnaJB11-specific IgG conjugated to rhodamine (DnaJB11-rhodamine, red). (J) Immunogold labeling of DnaJB11 on P7-1 tubules in Sf9 cells co-expressed with P7-1 and DnaJB11C. Cells expressed with DnaJB11 alone (I) or co-expressed with P7-1 and DnaJB11C (II) were immunolabeled with DnaJB11 antibodies and goat antibodies against rabbit IgG that had been conjugated with 10-nm-diameter gold particles (arrows) as secondary antibodies. (K) At 48 hpi, Sf9 cells triply-expressed with P7-1, BAP31 and DnaJB11C were fixed and immunolabeled with BAP31-FITC (green), P7-1-rhodamine (red) and DnaJB11-Alexa Fluor 647 (blue). BF, brightfield. Bars in A-I, K, 5 μm. Bar in J, 100 nm.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Immunolabeling, Staining, Incubation, Labeling

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A-B) The transcript levels of Sf-DnaJB11 (A) and Sf-BAP31 (B) in Sf9 cells infected with recombinant baculovirus containing P7-1 after treatment with dsGFP, dsSf-BAP31 or dsSf-DnaJB11 were detected by RT-qPCR assay. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in Sf-BAP31 or Sf-DnaJB11 transcript levels among S . furcifera treated with different dsRNAs. (C) Sf9 cells infected with recombinant baculovirus containing P7-1 after treatment with dsGFP, dsSf-BAP31 or dsSf-DnaJB11 were fixed at 18 or 36 hpi and immunolabeled with P7-1-FITC (green). BF, brightfield. Bars, 5 μm.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Infection, Recombinant, Quantitative RT-PCR, Immunolabeling

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A) Effects of SRBSDV infection on the transcript levels of BAP31 and DnaJB11 in viruliferous or nonviruliferous insects, as detected by RT-qPCR assay. Error bars indicate standard deviations from three biological replicates. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in BAP31 or DnaJB11 transcript levels between nonviruliferous and viruliferous S . furcifera . (B) Effects of SRBSDV infection on the protein levels of BAP31, DnaJB11, P9-1 or P7-1 in viruliferous or nonviruliferous insects, as detected by western blot assay by using BAP31-, DnaJB11-, P9-1- or P7-1-specific IgGs. Insect GAPDH was used as an internal control. (C) At 6 days padp, the midguts of nonviruliferous or viruliferous insects were immunolabeled with DnaJB11-FITC (green) and P7-1-rhodamine (red) and then examined by immunofluorescence microscopy. DnaJB11 and P7-1 tubules were co-localized in the midgut. mg, midgut. Bars, 30 μm. (D) Immunogold labeling of DnaJB11 on virus-containing tubules in SRBSDV-infected midgut from nonviruliferous (I) and viruliferous (II) insects. The midguts were immunolabled with DnaJB11 antibodies and goat antibodies against rabbit IgG that had been conjugated with 10-nm-diameter gold particles (arrows) as secondary antibodies. Bars, 100 nm. (E) At 6 days padp, the midguts of nonviruliferous or viruliferous insects were immunolabeled with BAP31-FITC (green) and P7-1-rhodamine (red) and then examined by immunofluorescence microscopy. Bars, 30 μm.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control, Immunolabeling, Immunofluorescence, Microscopy, Labeling, Virus

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A-D) The transcript levels of BAP31 (A), DnaJB11 (B), P9-1 (C), or P7-1 (D) from dsRNAs (dsGFP, dsBAP31 and dsDnaJB11)-treated or untreated viruliferous insects were detected by RT-qPCR assay. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in BAP31, DnaJB11, P9-1 or P7-1 transcript levels among viruliferous S . furcifera treated with different dsRNAs. (E) The protein levels of P9-1, P7-1, DnaJB11 or BAP31 from dsRNAs-treated virulifeorus insects were detected by western blot assay by using P9-1-, P7-1-, DnaJB11-, or BAP31-specific IgGs. Insect GAPDH was used as an internal control. (F) The visceral muscles of the midguts of viruliferous dsRNAs-treated and untreated insects were immunolabeled with P9-1-FITC (green), P7-1-rhodamine (red) and the actin dye phalloidin-Alexa Fluor 647 (blue), then examined by immunofluorescence microscopy. vm, visceral muscles. Bars, 30 μm. (G) Transmission rates of SRBSDV by individual viruliferous S . furcifera untreated or treated with different dsRNAs. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in transmission rates of SRBSDV by different dsRNAs-treated viruliferous S . furcifera . NT, untreated viruliferous insects.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Quantitative RT-PCR, Western Blot, Control, Muscles, Immunolabeling, Immunofluorescence, Microscopy, Transmission Assay

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A-B) The transcript levels of DnaJB11 (A) and BAP31 (B) in nonviruliferous or viruliferous insects after incubation with 15, 20, 25 and 35°C at 6 days padp were measured by RT-qPCR assay. Error bars indicate standard deviations from three biological replicates. Means (±SD) from three biological replicates are shown. Multiple comparisons of the means were conducted using a Tukey’s honest significant difference (HSD) test with a one-way analysis of variance (ANOVA). * P <0.05, ** P <0.01. (C) The protein levels of BAP31 or DnaJB11 in viruliferous insects after incubation with 15, 20, 25 and 35°C at 6 days padp were detected by western blot assay by using BAP31- or DnaJB11-specific IgGs. Insect GAPDH was detected with GAPDH-specific IgG as an internal control.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Incubation, Quantitative RT-PCR, Western Blot, Control

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: Two factors of ERAD machinery, DnaJB11 and BAP31 compete to interact with the tubule protein P7-1 of SRBSDV; however, DnaJB11 promotes whereas BAP31 inhibits P7-1 tubule assembly. Furthermore, BAP31 negatively regulates DnaJB11 expression through their direct interaction. Thus, high temperature can significantly upregulate DnaJB11 expression but inhibit BAP31 expression, finally facilitating the assembly of abundant P7-1 tubules.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Expressing