lman1 polyclonal antibody  (Bioss)

Journal: Journal of Virology

Article Title: Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway

doi: 10.1128/jvi.00005-22

Figure Lengend Snippet: Subcellular localization of TSG101 and PRRSV N protein, along with the early secretory pathway. (A) Subcellular localization of TSG101 with the early secretory pathway. The MARC-145 cells seeded in glass dishes were infected with or without PRRSV (MOI = 10) at 37°C for 10 h. The cells were costained with mouse anti-TSG101 MAb (green) and rabbit anti-PDI MAb (red), rabbit anti-LMAN1 pAbs (red), or rabbit anti-GM130 MAb (red), respectively. Nuclei were stained with DAPI (blue). Alexa Fluor 647-goat anti-rabbit IgG and Alexa Fluor 488-goat anti-mouse IgG were used as secondary antibodies. Images were taken at a 630× magnification and representative as a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 μm. The colocalization was assessed by determination of Manders’ colocalization coefficient using the JaCoP plugin in ImageJ software. The coefficient represents the proportion of the colocalized fluorescence intensity of A protein with B protein to total fluorescence intensity of A protein. The mean value ± the SEM is representative of three individual enlarged pictures. Statistical analysis was carried out using the Student t test. **, P < 0.01; ***, P < 0.001. (B) Interaction between TSG101 and PRRSV N protein, along with the early secretory pathway. The MARC-145 cells seeded in glass dishes were infected with PRRSV (MOI = 10). After incubation at 37°C for 10 h, the cells were first costained with mouse anti-PRRSV N MAb (green) and rabbit anti-PDI MAb (purple), rabbit anti-LMAN1 pAbs (purple), or rabbit anti-GM130 MAb (purple), respectively. Subsequently, the cells were costained with Alexa Fluor 647-conjugated TSG101 MAb (red), Alexa Fluor 555-goat anti-rabbit IgG, and Alexa Fluor 488-goat anti-mouse IgG antibodies. Nuclei were stained with DAPI (blue). Images were taken at a 630× magnification and representative as a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 μm. The colocalization was assessed by determination of Manders’ overlap coefficient using the JaCoP plugin in ImageJ software. The mean value ± the SEM is representative of three individual enlarged pictures.

Article Snippet: Rabbit anti-LMAN1 pAbs (catalog no. bs-18304R) was purchased from Bioss (Beijing, China).

Techniques: Infection, Staining, Software, Fluorescence, Incubation

Journal: Journal of Virology

Article Title: Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway

doi: 10.1128/jvi.00005-22

Figure Lengend Snippet: TSG101 knockdown affects the subcellular localization of PRRSV N protein with the early secretory pathway. (A) TSG101 knockdown impaired the subcellular distribution of PRRSV N protein. The MARC-145 cells seeded in glass dishes were transfected with either siTSG101 or siNC for 36 h and then infected with PRRSV (MOI = 10) at 37°C for 10 h. The cells were costained with mouse anti-PRRSV N MAb (green), rabbit anti-PDI MAb (red), rabbit anti-LMAN1 pAbs (red), or rabbit anti-GM130 MAb (red). Nuclei were stained with DAPI (blue). Alexa Fluor 647-goat anti-rabbit IgG and Alexa Fluor 488-goat anti-mouse IgG were used as secondary antibodies. Images were taken at a 630× magnification and representative as a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 μm. The colocalization was assessed by determination of Manders’ overlap coefficient using the JaCoP plugin in ImageJ software. The mean value ± the SEM is representative of three individual enlarged pictures. (B) IB analyses of ER/GA-enriched proteins. The MARC-145 cells seeded in 100-mm dishes were transfected with either siTSG101 or siNC for 36 h and infected with PRRSV (MOI = 10) at 37°C for 10 h. The cells were then collected and subjected to ER/GA protein extraction assay using a Minute ER/GA enrichment kit, respectively. The extracted proteins were subsequently examined by IB and stained with rabbit anti-TSG101 pAbs or rabbit anti-PRRSV N pAbs, along with rabbit anti-PDI MAb or rabbit anti-GM130 MAb as a loading control.

Article Snippet: Rabbit anti-LMAN1 pAbs (catalog no. bs-18304R) was purchased from Bioss (Beijing, China).

Techniques: Transfection, Infection, Staining, Software, Protein Extraction

Journal: Journal of Virology

Article Title: Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway

doi: 10.1128/jvi.00005-22

Figure Lengend Snippet: TSG101 is important for PRRSV infection in CRL-2843-CD163 cells. (A to C) Knockdown of TSG101 inhibited PRRSV infection. CRL-2843-CD163 cells were transfected with 50 nM siRNAs against pig TSG101 (siTSG101-320, siTSG101-494, and siTSG101-786) or siNC for 36 h and then inoculated with PRRSV (MOI = 0.5) at 37°C for additional 24 h. The infected cells were harvested for detection of PRRSV RNA abundance using RT-qPCR (A), endogenous TSG101 or PRRSV N protein expression using IB (B), and the extracellular progeny virus titers by detecting TCID 50 (C). (D) Cytotoxicities of the indicated siRNAs. The CRL-2843-CD163 cells grown in 96-well plates were transfected with the indicated siRNAs (50 nM) for 60 h, and the cell viabilities were evaluated by performing a CellTiter 96 AQueous One solution cell proliferation assay. The cell viability in siNC-transfected cells was set to 1.0. Data represent means ± the SEM from three independent experiments. Statistical analysis was carried out using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant ( P > 0.05). (E) Confocal microscopy analyses of the colocalization of PRRSV N protein with subcellular markers (PDI for ER, LMAN1 for ERGIC, and TGN46 for GA). The CRL-2843-CD163 cells seeded in glass dishes were transfected with either siTSG101-786 or siNC for 36 h and then infected with PRRSV (MOI = 20) at 37°C for 10 h. The cells were stained with mouse anti-PRRSV N MAb (green), rabbit anti-PDI MAb (red), rabbit anti-LMAN1 pAbs (red), or rabbit anti-TGN46 pAbs (red). Nuclei were stained with DAPI (blue). Alexa Fluor 647-goat anti-rabbit IgG and Alexa Fluor 488-goat anti-mouse IgG were used as secondary antibodies. Images were taken at a 630× magnification and are representative as a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 μm. The colocalization was assessed by determination of Manders’ overlap coefficient using the JaCoP plugin in ImageJ software. The mean value ± the SEM is representative of three individual enlarged pictures.

Article Snippet: Rabbit anti-LMAN1 pAbs (catalog no. bs-18304R) was purchased from Bioss (Beijing, China).

Techniques: Infection, Transfection, Quantitative RT-PCR, Expressing, Proliferation Assay, Confocal Microscopy, Staining, Software