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Santa Cruz Biotechnology 136172 rrid ab 2023156
136172 Rrid Ab 2023156, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+larg+antibody/MCPyV+large+T-antigen+Antibody/pmc13047360-77-11-7
Average 93 stars, based on 109 article reviews

136172 rrid ab 2023156 - by Bioz Stars, 2026-10

93/100 stars

Images

Centrifugation:

Article Title: Unc5B associates with LARG to mediate the action of repulsive guidance molecule
Article Snippet: The supernatants collected were precleared for 30 min by incubating with 60 μl protein A/G–Sepharose beads (GE Healthcare). .. After a brief centrifugation to remove the precleared beads, the cell lysates were incubated for 2 h (for co-IP with transfected 293T cell extracts) or overnight (for co-IP with rat cortical neuron extracts) at 4°C with anti–VSV-G antibody (Sigma-Aldrich), anti-HA antibody (Sigma-Aldrich), anti–c-myc antibody (Santa Cruz Biotechnology, Inc.), antineogenin (Santa Cruz Biotechnology, Inc.), anti-Unc5B (R&D Systems), or anti-LARG antibody (Santa Cruz Biotechnology, Inc.). ..

Incubation:

Transfection:

other:

Article Title: PDZ-RhoGEF ubiquitination by Cullin3–KLHL20 controls neurotrophin-induced neurite outgrowth
Article Snippet: The anti-LARG antibody was purchased from Santa Cruz Biotechnology, Inc., and anti-Cul3 antibodies were purchased from Santa Cruz Biotechnology, Inc. and Gene Tex.

Image Search Results

(a-a‴) Hemolymph from an undamaged larva containing circulating hemocytes expressing srpHemo-H2A::3XmCh (a′, a‴) and Lz-GFP (a″, a‴), co-stained with anti-Nimrod (a, a‴). The yellow arrowhead indicates a Lz-GFP-positive crystal cell, orange arrowheads indicate srpHemo-H2A::3XmCh-positive hemocytes, and white arrowheads indicate NimC-positive plasmatocytes. (b-b‴) Hemolymph from an undamaged larva expressing srpHemo::3XmCh co-stained with anti-Atilla and DAPI. Blue arrowheads indicate Atilla-positive lamellocytes co-expressing srpHemo::3XmCh. (c-f) Wing discs from larvae expressing srpHemo-H2A::3XmCh. (c) Undamaged control disc. (d) R0 disc. (e) R24 disc. (f) R48 disc. (g-g′) Wing disc from larva expressing srpHemo-H2A::3XmCh at R48 co-stained with anti-Antennapedia (Antp). Yellow arrowhead indicates srpHemo-H2A::3XmCh-positive cells. (h-h′) YZ orthogonal projections of R48 wing discs from larvae expressing srpHemo-H2A::3XmCh to mark putative immune cells and rn >EYFP to mark the cell debris co-stained with anti-Discs large (Dlg) to mark cell membranes and DAPI. Yellow arrowheads indicate srpHemo-H2A::3XmCh-positive cells within the regenerating epithelium. (i-i″) R48 wing disc from larvae co-expressing srpHemo-moe::3XmCh to mark membranes of putative immune cells and stained with anti-Nubbin (Nub) to mark the wing pouch and DAPI. Yellow arrowhead labels columnar epithelial pouch cells expressing both the srpHemo-moe::3XmCh reporter and Nubbin. Scale bars: 50 µm.

Journal: bioRxiv

Article Title: Draper-mediated efferocytosis by Drosophila imaginal disc epithelial cells clears cellular debris during regeneration

doi: 10.64898/2026.05.04.722804

Figure Lengend Snippet: (a-a‴) Hemolymph from an undamaged larva containing circulating hemocytes expressing srpHemo-H2A::3XmCh (a′, a‴) and Lz-GFP (a″, a‴), co-stained with anti-Nimrod (a, a‴). The yellow arrowhead indicates a Lz-GFP-positive crystal cell, orange arrowheads indicate srpHemo-H2A::3XmCh-positive hemocytes, and white arrowheads indicate NimC-positive plasmatocytes. (b-b‴) Hemolymph from an undamaged larva expressing srpHemo::3XmCh co-stained with anti-Atilla and DAPI. Blue arrowheads indicate Atilla-positive lamellocytes co-expressing srpHemo::3XmCh. (c-f) Wing discs from larvae expressing srpHemo-H2A::3XmCh. (c) Undamaged control disc. (d) R0 disc. (e) R24 disc. (f) R48 disc. (g-g′) Wing disc from larva expressing srpHemo-H2A::3XmCh at R48 co-stained with anti-Antennapedia (Antp). Yellow arrowhead indicates srpHemo-H2A::3XmCh-positive cells. (h-h′) YZ orthogonal projections of R48 wing discs from larvae expressing srpHemo-H2A::3XmCh to mark putative immune cells and rn >EYFP to mark the cell debris co-stained with anti-Discs large (Dlg) to mark cell membranes and DAPI. Yellow arrowheads indicate srpHemo-H2A::3XmCh-positive cells within the regenerating epithelium. (i-i″) R48 wing disc from larvae co-expressing srpHemo-moe::3XmCh to mark membranes of putative immune cells and stained with anti-Nubbin (Nub) to mark the wing pouch and DAPI. Yellow arrowhead labels columnar epithelial pouch cells expressing both the srpHemo-moe::3XmCh reporter and Nubbin. Scale bars: 50 µm.

Article Snippet: Primary antibodies used were mouse anti-cut (1:150, DSHB Cat# 2b10, RRID: AB_528186), mouse anti-Nimrod (1:250, gift from I. Ando) (Kurucz, Márkus, et al. 2007), mouse anti-AtillaL1abc (1:100, gift from I. Ando) (Kurucz, Váczi, et al. 2007), mouse anti-Lozenge (1:10, DSHB, RRID: AB_528346) , mouse anti-Hemese (1:100) (gift from I. Ando) , mouse anti-Antennapedia (1:10, DSHB Cat# 8C11, RRID: AB_528083), mouse anti-Discs large (1:100, DSHB Cat# 4F3, RRID: AB_528203), mouse anti-Nubbin (1:100, DSHB Cat# 2D4, RRID:AB_2722119), rabbit anti-Draper (1:500) (gift from M. Freeman) , mouse anti-Fibrillarin (1:100; Abcam, ab4566), mouse anti-Lamin (1:100, DSHB Cat# ADL67.10, RRID: AB_528336).

Techniques: Expressing, Staining, Control

(a-a’’) Orthogonal YZ projection of R10 wing disc expressing rn >EYFP with DAPI staining. Blue arrowheads indicate EYFP-labelled cell debris in the hinge epithelium. For all the panels, yellow arrowhead: Notum-Hinge fold (N-H); Orange arrowhead: Hinge-Hinge fold, purple arrowhead: Hinge-Pouch fold (H-P). (b, c) Top-down single-slice views of undamaged control and R0 wing discs stained with LysoTracker. Yellow dashed box-higher magnification region of interest (d-e) Higher-magnification views of the R0 disc in (c) (yellow dashed box) showing LysoTracker-positive puncta (d,d’,e’), anti-Discs large (Dlg) to show epithelial membranes (d’), and rn >EYFP-labeled debris (e,e’). Orange arrowhead: Hinge-Hinge fold, blue arrowheads indicate lysosomes. (f) Top-down single-slice view of an R24 wing disc stained with LysoTracker. (g) Quantification of lysosome number (LysoTracker-positive puncta) across regeneration time points. Undamaged control n=10 discs, R0 n=9 discs, ****P= 0.0001 compared to undamaged control, R24 n=11 discs, ****P=0.0001 compared to R0. (h-h‴) YZ orthogonal projection of R0 disc stained with LysoTracker (h,h’,h’’’), Dlg (h’), and expressing rn >EYFP to label debris (h’’). The white dotted circle marks lysosomes localized to the hinge-pouch (H-P) fold. (i-i‴) YZ orthogonal projection of R0 disc expressing rn >EYFP to mark the debris (i’) and TRE-RFP to mark JNK signaling (i’’,i’’’), and stained with LysoTracker (i,i’) and DAPI (i’’’). The yellow dotted circle marks lysosomes localized in the H-P fold that are associated with JNK signal activation. Error bars: SEM. Scale bars: 50 µm.

Journal: bioRxiv

Article Title: Draper-mediated efferocytosis by Drosophila imaginal disc epithelial cells clears cellular debris during regeneration

doi: 10.64898/2026.05.04.722804

Figure Lengend Snippet: (a-a’’) Orthogonal YZ projection of R10 wing disc expressing rn >EYFP with DAPI staining. Blue arrowheads indicate EYFP-labelled cell debris in the hinge epithelium. For all the panels, yellow arrowhead: Notum-Hinge fold (N-H); Orange arrowhead: Hinge-Hinge fold, purple arrowhead: Hinge-Pouch fold (H-P). (b, c) Top-down single-slice views of undamaged control and R0 wing discs stained with LysoTracker. Yellow dashed box-higher magnification region of interest (d-e) Higher-magnification views of the R0 disc in (c) (yellow dashed box) showing LysoTracker-positive puncta (d,d’,e’), anti-Discs large (Dlg) to show epithelial membranes (d’), and rn >EYFP-labeled debris (e,e’). Orange arrowhead: Hinge-Hinge fold, blue arrowheads indicate lysosomes. (f) Top-down single-slice view of an R24 wing disc stained with LysoTracker. (g) Quantification of lysosome number (LysoTracker-positive puncta) across regeneration time points. Undamaged control n=10 discs, R0 n=9 discs, ****P= 0.0001 compared to undamaged control, R24 n=11 discs, ****P=0.0001 compared to R0. (h-h‴) YZ orthogonal projection of R0 disc stained with LysoTracker (h,h’,h’’’), Dlg (h’), and expressing rn >EYFP to label debris (h’’). The white dotted circle marks lysosomes localized to the hinge-pouch (H-P) fold. (i-i‴) YZ orthogonal projection of R0 disc expressing rn >EYFP to mark the debris (i’) and TRE-RFP to mark JNK signaling (i’’,i’’’), and stained with LysoTracker (i,i’) and DAPI (i’’’). The yellow dotted circle marks lysosomes localized in the H-P fold that are associated with JNK signal activation. Error bars: SEM. Scale bars: 50 µm.

Techniques: Expressing, Staining, Control, Labeling, Activation Assay

(A) Experimental design. UBQLN2 P497H was expressed pan-neuronally using elav-GAL4. Parental flies were maintained on standard food supplemented with SR-0813 (1 µM; SR-1 or 10 µM; SR-10) or left untreated (UNT). Progeny were assessed at the third-instar larval stage (L3) by crawling assays, and adults were assayed for climbing performance every 5 days from day 5 to day 30 post - eclosion (D5-D30). (B–E) Larval crawling behavior. (B) Representative 30-s locomotor tracks from L3 foraging larvae (UNT, SR-1, SR-10). Quantification of (C) track length, (D) distance traveled, and (E) average speed over 30 s. Each point represents one larva (UNT n = 60; SR-1 n = 53; SR-10 n = 57). Statistical analysis: ordinary one-way ANOVA with Dunnett’s multiple-comparisons test (each SR-0813 condition versus UNT); p < 0.05 was considered significant. (F–H) Neuromuscular junction (NMJ) morphology. (F) Representative images of type Ib NMJs on muscle 4 (abdominal segment A4) from L3 elav>UBQLN2 P497H larvae reared on UNT, SR-1, or SR-10 food. Presynaptic terminals were labeled with anti-HRP (green) and postsynaptic densities with anti-Discs large (Dlg; red). Quantification of (G) NMJ branch length and (H) bouton number. Each point represents one larva (n = 30 larvae per condition). Statistical analysis: ordinary one-way ANOVA with Dunnett’s multiple-comparisons test (each SR-0813 condition versus UNT); p < 0.05 was considered significant.

Journal: bioRxiv

Article Title: A leukemia-derived ENL/AF9 chemical probe enhances neuronal stress resilience and ameliorates ALS phenotypes

doi: 10.64898/2026.04.09.717610

Figure Lengend Snippet: (A) Experimental design. UBQLN2 P497H was expressed pan-neuronally using elav-GAL4. Parental flies were maintained on standard food supplemented with SR-0813 (1 µM; SR-1 or 10 µM; SR-10) or left untreated (UNT). Progeny were assessed at the third-instar larval stage (L3) by crawling assays, and adults were assayed for climbing performance every 5 days from day 5 to day 30 post - eclosion (D5-D30). (B–E) Larval crawling behavior. (B) Representative 30-s locomotor tracks from L3 foraging larvae (UNT, SR-1, SR-10). Quantification of (C) track length, (D) distance traveled, and (E) average speed over 30 s. Each point represents one larva (UNT n = 60; SR-1 n = 53; SR-10 n = 57). Statistical analysis: ordinary one-way ANOVA with Dunnett’s multiple-comparisons test (each SR-0813 condition versus UNT); p < 0.05 was considered significant. (F–H) Neuromuscular junction (NMJ) morphology. (F) Representative images of type Ib NMJs on muscle 4 (abdominal segment A4) from L3 elav>UBQLN2 P497H larvae reared on UNT, SR-1, or SR-10 food. Presynaptic terminals were labeled with anti-HRP (green) and postsynaptic densities with anti-Discs large (Dlg; red). Quantification of (G) NMJ branch length and (H) bouton number. Each point represents one larva (n = 30 larvae per condition). Statistical analysis: ordinary one-way ANOVA with Dunnett’s multiple-comparisons test (each SR-0813 condition versus UNT); p < 0.05 was considered significant.

Article Snippet: For immunostaining, neuromuscular junctions were labeled using FITC-conjugated goat anti-horseradish peroxidase (HRP) antibody (1:1000, Jackson ImmunoResearch), which marks neuronal membranes, and mouse anti-Discs large (Dlg) antibody (1:300, clone 4F3, DSHB).

Techniques: Labeling

(A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, SDS Page, Western Blot, Infection, Staining, Software, Construct, Expressing

$(A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3+ signal on or proximal to (i.e. within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with Bap31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in (). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. ( E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from fractions 1-7 (representing disassembled virus) and fraction 8 (representing assembled virus) was quantified and normalized to the Scr control. ** p ≤ 0.01; ns= not significant.$

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3+ signal on or proximal to (i.e. within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with Bap31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in (). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. ( E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from fractions 1-7 (representing disassembled virus) and fraction 8 (representing assembled virus) was quantified and normalized to the Scr control. ** p ≤ 0.01; ns= not significant.

Techniques: Transfection, Control, Infection, Staining, Membrane, Transport Assay, SDS Page, Western Blot, Virus

(A) Endogenous KPNA4 was IPed from whole cell extracts derived from SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting. (B) As in A, except CV-1 cells were subjected to SUN1 KD. (C) CV-1 cells transfected with the indicated siRNA and SUN1-3xFLAG were infected with SV40. SUN1-3xFLAG was IPed from the resulting whole cell extracts, and the precipitated samples subjected to SDS-PAGE and immunoblotting. (D) The VP1 band intensity in C was quantified by the FIJI software. Data were normalized to the Scr condition. (E) As in , except cells were depleted of KPNA4 and stained for FLAG (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Manders’ coefficient was used to quantify overlap of VP2/3+ signal on SUN1-3xFLAG. Each data point represents one cell. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. Cells were fixed and stained for mAb414 (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (H) The percent of cells with a discrete VP2/3+ signal in the nucleus in G was quantified and normalized to the Scr control condition. (I) Model: After SV40 reaches the cytosol from the ER (foci), the viral particle is partially disassembled and targeted to the Nesprin-2-SUN1 nuclear membrane protein complex (Step 1). The virus is then released to the KPNA4 importin α receptor, which translocates SV40 into the nucleus to promote infection (Step 2). Schematic created in biorender.com. * p ≤ 0.05, ** p ≤ 0.01

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Endogenous KPNA4 was IPed from whole cell extracts derived from SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting. (B) As in A, except CV-1 cells were subjected to SUN1 KD. (C) CV-1 cells transfected with the indicated siRNA and SUN1-3xFLAG were infected with SV40. SUN1-3xFLAG was IPed from the resulting whole cell extracts, and the precipitated samples subjected to SDS-PAGE and immunoblotting. (D) The VP1 band intensity in C was quantified by the FIJI software. Data were normalized to the Scr condition. (E) As in , except cells were depleted of KPNA4 and stained for FLAG (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Manders’ coefficient was used to quantify overlap of VP2/3+ signal on SUN1-3xFLAG. Each data point represents one cell. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. Cells were fixed and stained for mAb414 (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (H) The percent of cells with a discrete VP2/3+ signal in the nucleus in G was quantified and normalized to the Scr control condition. (I) Model: After SV40 reaches the cytosol from the ER (foci), the viral particle is partially disassembled and targeted to the Nesprin-2-SUN1 nuclear membrane protein complex (Step 1). The virus is then released to the KPNA4 importin α receptor, which translocates SV40 into the nucleus to promote infection (Step 2). Schematic created in biorender.com. * p ≤ 0.05, ** p ≤ 0.01

Techniques: Derivative Assay, Infection, SDS Page, Western Blot, Transfection, Software, Staining, Control, Membrane, Virus

(A) Endogenous SUN1 was immunoprecipitated (IPed) from whole cell extracts derived from SV40-infected CV-1 cells. Bound DNA was eluted from the precipitated material and subjected to PCR to detect the SV40 genomic DNA. (B) CV-1 cells expressing the indicated FLAG-tagged constructs were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts, the precipitated samples eluted and subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. * indicates degraded SUN proteins. (C) As in B, except the precipitated samples were subjected to PCR to detect the SV40 genome. (D) Nesprin-2 was IPed from whole cell extracts derived from CV-1 cells expressing the indicated constructs. The precipitated samples were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. * indicates degraded SUN proteins (E) As in B, except SUN1 ΔLU-3xFLAG was also used. (F) CV-1 cells transfected with the indicated siRNA and construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts and processed as in B. * indicates degraded SUN1 protein. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. The resulting whole cell extracts were subjected to Nesprin-2 IP and the precipitated samples subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (H) Endogenous BicD2 was IPed from whole cell extracts derived from CV-1 cells and the precipitated samples subjected to SDS-PAGE and immunoblotting. (I) Endogenous BicD2 was IPed from whole cell extracts derived from mock or SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting.

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Endogenous SUN1 was immunoprecipitated (IPed) from whole cell extracts derived from SV40-infected CV-1 cells. Bound DNA was eluted from the precipitated material and subjected to PCR to detect the SV40 genomic DNA. (B) CV-1 cells expressing the indicated FLAG-tagged constructs were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts, the precipitated samples eluted and subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. * indicates degraded SUN proteins. (C) As in B, except the precipitated samples were subjected to PCR to detect the SV40 genome. (D) Nesprin-2 was IPed from whole cell extracts derived from CV-1 cells expressing the indicated constructs. The precipitated samples were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. * indicates degraded SUN proteins (E) As in B, except SUN1 ΔLU-3xFLAG was also used. (F) CV-1 cells transfected with the indicated siRNA and construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts and processed as in B. * indicates degraded SUN1 protein. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. The resulting whole cell extracts were subjected to Nesprin-2 IP and the precipitated samples subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (H) Endogenous BicD2 was IPed from whole cell extracts derived from CV-1 cells and the precipitated samples subjected to SDS-PAGE and immunoblotting. (I) Endogenous BicD2 was IPed from whole cell extracts derived from mock or SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting.

Techniques: Immunoprecipitation, Derivative Assay, Infection, Expressing, Construct, SDS Page, Western Blot, Transfection

(A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP+Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP+Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01

Techniques: Transfection, Construct, Staining, Infection, Expressing, Control, SDS Page, Western Blot

(A) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE followed by immunoblotting. The extent of depletion by the KPNA1, KPNA2, and KPNA4 siRNA is shown in the Western blots below. Data were normalized to the Scr control. (B) The T-Ag band intensity in A was quantified by the FIJI software. Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) CV-1 cells were fixed and stained for Nesprin-2 (red) and either one of the KPNA1, KPNA2, or KPNA4 protein (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Pearson’s coefficient was used to quantify colocalization between KPNA1, KPNA2, or KPNA4 with Nesprin-2. Each data point represents one field of view with at least 10 cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE followed by immunoblotting. The extent of depletion by the KPNA1, KPNA2, and KPNA4 siRNA is shown in the Western blots below. Data were normalized to the Scr control. (B) The T-Ag band intensity in A was quantified by the FIJI software. Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) CV-1 cells were fixed and stained for Nesprin-2 (red) and either one of the KPNA1, KPNA2, or KPNA4 protein (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Pearson’s coefficient was used to quantify colocalization between KPNA1, KPNA2, or KPNA4 with Nesprin-2. Each data point represents one field of view with at least 10 cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Techniques: Transfection, Infection, SDS Page, Western Blot, Control, Software, Staining

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136172 rrid ab 2023156 (Santa Cruz Biotechnology)

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