mouse anti igg magnetic bead conjugate  (Cell Signaling Technology Inc)

Journal: Journal of Virology

Article Title: Autographa californica multiple nucleopolyhedrovirus Ac51 interacts with Ac66 and facilitates its nuclear localization to promote the nuclear egress of nucleocapsids

doi: 10.1128/jvi.01969-24

Figure Lengend Snippet: Effect of different domain deletion on the expression and localization of Ac51 in infected cells and virions. ( A ) Expression of truncated Ac51 in infected cells by western blot analysis. Sf9 cells were transfected with 1 µg of bacmid DNA of vAcWT, vAc51ΔDnaJ, vAc51ΔRRM, vAc51ΔCC, or vAc51KO. Cells were collected at 24 and 96 h p.t. for western blot analysis. Truncated Ac51 was detected using an anti-HA antibody, and GP64 was detected as the loading control. ( B ) Expression of truncated Ac51 in infected cells at 72 h p.t. by an immunofluorescence assay. Sf9 cells transfected with 1 µg of bacmid DNA of vAcWT, vAc51ΔDnaJ, vAc51ΔRRM, or vAc51ΔCC were fixed at 72 h p.t. The cells were probed with the anti-HA antibody, followed by a secondary antibody conjugated with Alexa Fluor Plus 555. Hoechst 33342 was used for nuclear staining. Bright field (BF) images were also captured. ( C ) The fluorescence intensity of truncated Ac51 in ( B ) was quantitatively analyzed. ImageJ software was used to analyze the fluorescence intensity of truncated Ac51 in nine randomly selected fields. The same analysis was performed on five randomly selected fields in vAcWT-infected cells. ( D ) The ratio of fluorescent cells to infected cells in ( B ) was determined in the nine randomly selected fields. ( E ) Localization of truncated Ac51 in BVs. BVs of the recombinant viruses were purified from the supernatant and analyzed by western blot analysis. Two nonspecific protein bands were indicated by asterisks.

Article Snippet: One part was incubated with 15 μL of IgG-conjugated magnetic beads, and the other part was incubated with 15 μL of Flag-conjugated magnetic beads (P2115, Beyotime) with rotation at 4°C overnight.

Techniques: Expressing, Infection, Western Blot, Transfection, Control, Immunofluorescence, Staining, Fluorescence, Software, Recombinant, Purification

Journal: Journal of Virology

Article Title: Autographa californica multiple nucleopolyhedrovirus Ac51 interacts with Ac66 and facilitates its nuclear localization to promote the nuclear egress of nucleocapsids

doi: 10.1128/jvi.01969-24

Figure Lengend Snippet: Effect of ac51 deletion on the nuclear import of Ac66. ( A ) Subcellular distribution of Ac66 in vAc51KO- or vAcWT-infected cells at 72 h p.t. Sf9 cells were co-transfected with pUC18-Ac66Flag and bacmid DNA of vAc51KO or vAcWT and fixed at 36 h p.t. and 72 h p.t. Ac66 was detected with the anti-Flag antibody, followed by probing with the secondary antibody conjugated with Alexa Fluor Plus 647. ( B ) Different distribution pattern of Ac66 in vAc51KO-infected cells at 72 h p.t. was shown. ( C and D ) The nuclear proportion of Ac66 was analyzed at 36 h p.t. ( C ) and 72 h p.t. ( D ). ( E and F ) Fluorescence intensity of Ac66 was analyzed at 36 h p.t. ( E ) and 72 h p.t. ( F ). Infected cells were randomly selected, and nuclear proportion and the total amount of Ac66 in the whole cell were analyzed by measuring the fluorescence intensity of Ac66 using ImageJ software. The number of cells analyzed ( N ) was 16 and 21 for vAc51KO and vAcWT at 36 h p.t., respectively, and n was 21 and 22 for vAc51KO and vAcWT at 72 h p.t.

Article Snippet: One part was incubated with 15 μL of IgG-conjugated magnetic beads, and the other part was incubated with 15 μL of Flag-conjugated magnetic beads (P2115, Beyotime) with rotation at 4°C overnight.

Techniques: Infection, Transfection, Fluorescence, Software

Journal: Journal of Virology

Article Title: Autographa californica multiple nucleopolyhedrovirus Ac51 interacts with Ac66 and facilitates its nuclear localization to promote the nuclear egress of nucleocapsids

doi: 10.1128/jvi.01969-24

Figure Lengend Snippet: Interaction analysis between truncated Ac51 with Ac66 determined by Co-IP and immunofluorescence assays. ( A ) Interaction analysis between truncated Ac51 with Ac66 was performed using Co-IP assay. Sf9 cells were co-transfected with bacmid DNA of vAc51ΔDnaJ, vAc51ΔRRM, or vAc51ΔCC, along with pUC18-Ac66Flag, and collected at 90 h p.t. Additionally, Sf9 cells co-transfected with bacmid DNA of vAc51CHA and pUC18-Ac66Flag were collected at 48 h p.t. The cell lysate was subjected to a Co-IP assay with anti-Flag or IgG magnetic beads. Asterisks were used to indicate non-specific protein bands. ( B ) Colocalization of truncated Ac51 with Ac66 in infected cells. Sf9 cells were treated similarly to the Co-IP assay and fixed at 72 h p.t. Ac51CHA was probed with an anti-Ac51 antibody, while truncated Ac51 was probed with an anti-HA antibody. Ac66 was probed with an anti-Flag antibody. Cells were then incubated with the goat anti-mouse secondary antibody conjugated with Alexa Fluor Plus 647 or the goat anti-rabbit secondary antibody conjugated with Alexa Fluor Plus 555. ( C ) PCC between truncated Ac51 and Ac66. Randomly selected infected cells (vAc51CHA, n = 23; vAc51ΔDnaJ, n = 20; vAc51ΔRRM, n = 21; vAc51ΔCC, n = 59) were analyzed using ImageJ software, which calculates the PCC between proteins. ( D ) The nuclear proportion of Ac66 in vAc51ΔDnaJ-, vAc51ΔRRM-, or vAc51ΔCC-infected cells. Randomly selected infected cells (vAc51CHA, n = 23; vAc51ΔDnaJ, n = 17; vAc51ΔRRM, n = 23; vAc51ΔCC, n = 54) were analyzed using ImageJ software to determine the ratio of nuclear proportion vs whole cells.

Article Snippet: One part was incubated with 15 μL of IgG-conjugated magnetic beads, and the other part was incubated with 15 μL of Flag-conjugated magnetic beads (P2115, Beyotime) with rotation at 4°C overnight.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Transfection, Magnetic Beads, Infection, Incubation, Software

Journal: Journal of Virology

Article Title: Autographa californica multiple nucleopolyhedrovirus Ac51 interacts with Ac66 and facilitates its nuclear localization to promote the nuclear egress of nucleocapsids

doi: 10.1128/jvi.01969-24

Figure Lengend Snippet: Interaction and colocalization analysis between ME53 and Ac66, ME53 and Ac51, Ac132 and Ac51, and Ac132 and Ac66. ( A and B ) Interaction between ME53 and Ac51/Ac66 by Co-IP assay. Sf9 cells were transfected with pIB-ME53GFP. At 24 h p.t., cells were infected with vAcWT ( A ) or vAcWT Ac66flag ( B ) at a multiplicity of infection (MOI) of 5 TCID 50 /mL. At 48 h p.i., the cells were collected and subjected to the Co-IP assay using the anti-Ac51 antibody ( A ) or anti-Flag antibody ( B ). IgG from correspondent species was used as the negative control. All samples from the Co-IP assay were analyzed by western blot analysis with the respective antibodies indicated in the figures. ( C and D ) Interaction analysis between Ac132 and Ac51/Ac66 by Co-IP assay. Sf9 cells infected with vAc132HA ( C ) or co-infected with vAc132HA and vAcWT Ac66flag ( D ) at an MOI of 5 TCID 50 /mL were collected at 48 h p.i. and subjected to Co-IP assay with the anti-Ac51 antibody ( C ) or anti-Flag antibody ( D ). IgG from correspondent species was used as the negative control. All samples from the Co-IP assay were analyzed by western blot analysis using the respective antibodies indicated in the figures. ( E and F ) Colocalization analysis between Ac132 and Ac51 ( F ) or between Ac132 and Ac66 ( F ). Sf9 cells treated the same as in the Co-IP assay were subjected to the immunofluorescence assay at 48 h p.i. Ac132, Ac51, and Ac66 were probed with the anti-HA, anti-Ac51, and anti-Flag antibodies, respectively. ( G and H ) Colocalization analysis between ME53 and Ac51 ( G ) or between ME53 and Ac66 ( H ). Sf9 cells co-transfected with pUC18-ME53Myc and bacmid DNA of vAcWT ( G ) or vAcWT Ac66Flag ( H ) were subjected to the immunofluorescent assay at 48 h p.t. ME53, Ac51, and Ac66 were probed with the anti-Myc, anti-Ac51, and anti-Flag antibodies, respectively.

Article Snippet: One part was incubated with 15 μL of IgG-conjugated magnetic beads, and the other part was incubated with 15 μL of Flag-conjugated magnetic beads (P2115, Beyotime) with rotation at 4°C overnight.

Techniques: Co-Immunoprecipitation Assay, Transfection, Infection, Negative Control, Western Blot, Immunofluorescence