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ti e yokogawa csu w1 nikon c2 inverted confocal  (Yokogawa Electric)
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Yokogawa Electric ti e yokogawa csu w1 nikon c2 inverted confocal
Ti E Yokogawa Csu W1 Nikon C2 Inverted Confocal, supplied by Yokogawa Electric, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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digital eclipse nikon c1 confocal laser scanning microscope clsm  (Nikon)
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Nikon digital eclipse nikon c1 confocal laser scanning microscope clsm
Digital Eclipse Nikon C1 Confocal Laser Scanning Microscope Clsm, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti β actin clone c 2 abs  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology anti β actin clone c 2 abs
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Anti β Actin Clone C 2 Abs, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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eclipse c2 si confocal spectral microscope  (Nikon)
99
Nikon eclipse c2 si confocal spectral microscope
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Eclipse C2 Si Confocal Spectral Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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laser scanning confocal microscope  (Nikon)
99
Nikon laser scanning confocal microscope
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Laser Scanning Confocal Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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eclipse ti2 c2 confocal microscope  (Nikon)
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Nikon eclipse ti2 c2 confocal microscope
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Eclipse Ti2 C2 Confocal Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lsm 880 confocal microscope  (Carl Zeiss)
90
Carl Zeiss lsm 880 confocal microscope
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Lsm 880 Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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fluorescence microscope  (Olympus)
99
Olympus fluorescence microscope
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Fluorescence Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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fluorescence microscope - by Bioz Stars, 2026-06
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lsm780 confocal microscope system  (Carl Zeiss)
90
Carl Zeiss lsm780 confocal microscope system
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Lsm780 Confocal Microscope System, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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tie inverted microscope  (Nikon)
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Nikon tie inverted microscope
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
Tie Inverted Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a1 confocal microscopes  (Nikon)
96
Nikon a1 confocal microscopes
Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with <t>anti-β-actin</t> antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).
A1 Confocal Microscopes, supplied by Nikon, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti vangl2 c 2  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology anti vangl2 c 2
( A and B ) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) (A) and immunoblotting (B) analysis of <t>VANGL2</t> mRNA and protein level change in A549 cells infected with vesicular stomatitis virus (VSV) [multiplicity of infection (MOI) of 0.5] for 0 to 16 hours. ( C ) Immunoblotting analysis of VANGL2 protein level changes in wild-type (WT) and Ifnar −/− peritoneal macrophages (PEMs) infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Luciferase reporter assays analyzing IFN-β or IFN-stimulated response element (ISRE) promoter activity of human embryonic kidney (HEK) 293T cells transfected with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 or empty vector (EV) for 24 hours, followed by treatment with or without VSV (MOI of 0.5) (D) or poly(I:C) (E) for 12 hours, respectively. ( F to J ) Immunoblotting analysis (F and I) of total and phosphorylated IRF3 and RT-PCR analysis (G), (H), and (J) of indicated gene expression in HEK293T (F) to (H) or A549 (I) and (J) cells transfected with FLAG-VANGL2 or EV for 24 hours, followed by VSV (MOI of 0.5) infection at indicated time points. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( K and L ) Fluorescence microscopy analysis (K) and flow cytometric analysis (L) of the replication of VSV–enhanced green fluorescent protein (eGFP) in HEK293T cells transfected with EV or increasing HA-VANGL2 at indicated dose for 24 hours, followed by treatment with or without VSV-eGFP (MOI of 0.5) infection at indicated time points. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. NC, negative control. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test. ns, not significant.
Anti Vangl2 C 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with anti-β-actin antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).

Journal: Oncotarget

Article Title: Discoidin domain receptor 1 promotes Th17 cell migration by activating the RhoA/ROCK/MAPK/ERK signaling pathway.

doi: 10.18632/oncotarget.10455

Figure Lengend Snippet: Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with anti-β-actin antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).

Article Snippet: Non-conjugated rabbit anti-mouse and human DDR1 (clone C-20), non-conjugated rabbit anti-human DDR2 (clone H108) antibodies and the anti-phospho-ERK1/2 (clone E-4), anti-ERK2 (clone C-14), and anti-β-actin (clone C-2) Abs were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Transfection, Control, Western Blot, Staining, Flow Cytometry, Derivative Assay, Migration, Confocal Microscopy, Cell Tracking Assay, Recombinant

( A and B ) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) (A) and immunoblotting (B) analysis of VANGL2 mRNA and protein level change in A549 cells infected with vesicular stomatitis virus (VSV) [multiplicity of infection (MOI) of 0.5] for 0 to 16 hours. ( C ) Immunoblotting analysis of VANGL2 protein level changes in wild-type (WT) and Ifnar −/− peritoneal macrophages (PEMs) infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Luciferase reporter assays analyzing IFN-β or IFN-stimulated response element (ISRE) promoter activity of human embryonic kidney (HEK) 293T cells transfected with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 or empty vector (EV) for 24 hours, followed by treatment with or without VSV (MOI of 0.5) (D) or poly(I:C) (E) for 12 hours, respectively. ( F to J ) Immunoblotting analysis (F and I) of total and phosphorylated IRF3 and RT-PCR analysis (G), (H), and (J) of indicated gene expression in HEK293T (F) to (H) or A549 (I) and (J) cells transfected with FLAG-VANGL2 or EV for 24 hours, followed by VSV (MOI of 0.5) infection at indicated time points. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( K and L ) Fluorescence microscopy analysis (K) and flow cytometric analysis (L) of the replication of VSV–enhanced green fluorescent protein (eGFP) in HEK293T cells transfected with EV or increasing HA-VANGL2 at indicated dose for 24 hours, followed by treatment with or without VSV-eGFP (MOI of 0.5) infection at indicated time points. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. NC, negative control. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test. ns, not significant.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) (A) and immunoblotting (B) analysis of VANGL2 mRNA and protein level change in A549 cells infected with vesicular stomatitis virus (VSV) [multiplicity of infection (MOI) of 0.5] for 0 to 16 hours. ( C ) Immunoblotting analysis of VANGL2 protein level changes in wild-type (WT) and Ifnar −/− peritoneal macrophages (PEMs) infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Luciferase reporter assays analyzing IFN-β or IFN-stimulated response element (ISRE) promoter activity of human embryonic kidney (HEK) 293T cells transfected with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 or empty vector (EV) for 24 hours, followed by treatment with or without VSV (MOI of 0.5) (D) or poly(I:C) (E) for 12 hours, respectively. ( F to J ) Immunoblotting analysis (F and I) of total and phosphorylated IRF3 and RT-PCR analysis (G), (H), and (J) of indicated gene expression in HEK293T (F) to (H) or A549 (I) and (J) cells transfected with FLAG-VANGL2 or EV for 24 hours, followed by VSV (MOI of 0.5) infection at indicated time points. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( K and L ) Fluorescence microscopy analysis (K) and flow cytometric analysis (L) of the replication of VSV–enhanced green fluorescent protein (eGFP) in HEK293T cells transfected with EV or increasing HA-VANGL2 at indicated dose for 24 hours, followed by treatment with or without VSV-eGFP (MOI of 0.5) infection at indicated time points. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. NC, negative control. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test. ns, not significant.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Negative Control

( A ) Luciferase activity in HEK293T cells transfected with scrambled ( Scr ) small interfering RNA (siRNA) or siRNA-targeting VANGL2 for 24 hours and then transfected with an IFN-β luciferase (IFN-β luc) for 24 hours, followed by treatment with or without VSV (MOI of 0.5), poly(I:C), or poly (dA:dT) for 12 hours. ( B and C ) RT-PCR (B) and immunoblotting (C) analysis of VSV (MOI of 0.5)–infected THP-1 cells transfected with Scr siRNA or VANGL2 -specific siRNA at indicated time points. ( D ) Heatmap view of top and bottom gene list of RNA-sequence data sets. Microarray analysis for total RNA was performed for Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + bone marrow–derived macrophages (BMDMs) with or without VSV infection. ( E ) VANGL2 regulates antiviral response-relevant target genes, presented as a volcano plot of genes with differential expression after VSV infection in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs. FC, fold change. ( F ) Gene ontology (GO) enrichment analysis of the VANGL2-dependent genes in (E) (−log 2 P values). ( G ) Heatmap showing the change of indicated ISGs in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs with or without VSV infection. ( H to L ) RT-PCR analysis of Ifnb (H), Isg56 (I), and VSV-G (K) mRNA expression, IFN-β enzyme-linked immunosorbent assay (ELISA) (J), and immunoblotting (L) analysis of total and phosphorylated IRF3 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A ) Luciferase activity in HEK293T cells transfected with scrambled ( Scr ) small interfering RNA (siRNA) or siRNA-targeting VANGL2 for 24 hours and then transfected with an IFN-β luciferase (IFN-β luc) for 24 hours, followed by treatment with or without VSV (MOI of 0.5), poly(I:C), or poly (dA:dT) for 12 hours. ( B and C ) RT-PCR (B) and immunoblotting (C) analysis of VSV (MOI of 0.5)–infected THP-1 cells transfected with Scr siRNA or VANGL2 -specific siRNA at indicated time points. ( D ) Heatmap view of top and bottom gene list of RNA-sequence data sets. Microarray analysis for total RNA was performed for Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + bone marrow–derived macrophages (BMDMs) with or without VSV infection. ( E ) VANGL2 regulates antiviral response-relevant target genes, presented as a volcano plot of genes with differential expression after VSV infection in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs. FC, fold change. ( F ) Gene ontology (GO) enrichment analysis of the VANGL2-dependent genes in (E) (−log 2 P values). ( G ) Heatmap showing the change of indicated ISGs in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs with or without VSV infection. ( H to L ) RT-PCR analysis of Ifnb (H), Isg56 (I), and VSV-G (K) mRNA expression, IFN-β enzyme-linked immunosorbent assay (ELISA) (J), and immunoblotting (L) analysis of total and phosphorylated IRF3 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Sequencing, Microarray, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

( A and B ) Luciferase reporter assays analyzing IFN-β (A) or ISRE (B) promoter activity of HEK293T cells transfected with the Flag-tagged indicated plasmids along with EV or increasing amounts (from 100 to 200 ng) of HA-VANGL2. ( C ) Co-immunoprecipitation (co-IP; with anti-FLAG) and immunoblotting analysis using protein lysates of HEK293T cells transfected with indicated plasmids. WCL, whole cell lysates. ( D ) Co-IP (with anti-TBK1) and immunoblotting analysis using endogenous proteins lysates of control and VSV (MOI of 0.5, 12 hours)–infected BMDMs. ( E ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. ( F ) Quantitative analysis of the colocalization in (E). ( G ) Co-IP (with anti-VANGL2) and immunoblotting analysis using unsorted, cytosolic, and membrane lysates of THP-1 cells with or without VSV infection for 12 hours. ( H ) Schematic mapping of VANGL2. ( I ) Co-IP and immunoblotting analysis using lysates from HEK293T cells transfected with MYC-VANGL2 and its truncations along with FLAG-TBK1. ( J ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with vectors for HA-PKBD along with FLAG-TBK1. PkBD, the Prickle-binding domain. ( K and L ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with MYC-VANGL2 and its deletions along with FLAG-TBK1 (K) or infected with VSV (L) (MOI of 0.5). ( M and N ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with increasing amounts (from 300 to 500 ng) of HA-PKBD or EV along with FLAG-TBK1 (M) or infected with VSV (N) (MOI of 0.5). Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Luciferase reporter assays analyzing IFN-β (A) or ISRE (B) promoter activity of HEK293T cells transfected with the Flag-tagged indicated plasmids along with EV or increasing amounts (from 100 to 200 ng) of HA-VANGL2. ( C ) Co-immunoprecipitation (co-IP; with anti-FLAG) and immunoblotting analysis using protein lysates of HEK293T cells transfected with indicated plasmids. WCL, whole cell lysates. ( D ) Co-IP (with anti-TBK1) and immunoblotting analysis using endogenous proteins lysates of control and VSV (MOI of 0.5, 12 hours)–infected BMDMs. ( E ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. ( F ) Quantitative analysis of the colocalization in (E). ( G ) Co-IP (with anti-VANGL2) and immunoblotting analysis using unsorted, cytosolic, and membrane lysates of THP-1 cells with or without VSV infection for 12 hours. ( H ) Schematic mapping of VANGL2. ( I ) Co-IP and immunoblotting analysis using lysates from HEK293T cells transfected with MYC-VANGL2 and its truncations along with FLAG-TBK1. ( J ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with vectors for HA-PKBD along with FLAG-TBK1. PkBD, the Prickle-binding domain. ( K and L ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with MYC-VANGL2 and its deletions along with FLAG-TBK1 (K) or infected with VSV (L) (MOI of 0.5). ( M and N ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with increasing amounts (from 300 to 500 ng) of HA-PKBD or EV along with FLAG-TBK1 (M) or infected with VSV (N) (MOI of 0.5). Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Infection, Labeling, Confocal Microscopy, Binding Assay

( A and B ) RT-qPCR (A) and immunoblotting (B) analysis of TBK1 mRNA and protein level change in HEK293T cells transfected with HA-TBK1, and increasing amounts of MYC-VANGL2. ( C ) Immunoblotting analysis of total and phosphorylated TBK1 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Immunoblotting analysis of total and phosphorylated TBK1 using THP-1 (D) or PBMCs (E) transfected with Scr siRNA or VANGL2 siRNA for 24 hours and then infected with VSV (MOI of 0.5) for indicated time. ( F ) Immunoblotting analysis of protein extracts of HEK293T cells treated with CHX (100 μg/ml) for 12 hours, followed by treatment with MG132 (10 μM), Baf A1 (0.2 μM), or both for 6 hours. ( G ) Immunoblotting analysis of HEK293T cells transfected with indicated plasmids for 24 hours, followed by treatment with MG132 (10 μM), 3-MA (10 mM), or Baf A1 (0.2 μM) for 6 hours. ( H ) Immunoblotting analysis using lysates from HEK293T cells transfected with FLAG-VANGL2 or FLAG-EV for 24 hours, followed by treatment with rapamycin (250 nM) for indicated time. ( I ) Immunoblotting analysis using lysates from Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs treated with EBSS for indicated time. ( J ) Immunoblotting analysis of WT, BECLIN -KO, or ATG5 -KO HEK293T cells transfected with indicated plasmids. ( K ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated specific antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; violet, LAMP1 signal; blue, DAPI. Scale bars, 20 μm. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) RT-qPCR (A) and immunoblotting (B) analysis of TBK1 mRNA and protein level change in HEK293T cells transfected with HA-TBK1, and increasing amounts of MYC-VANGL2. ( C ) Immunoblotting analysis of total and phosphorylated TBK1 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Immunoblotting analysis of total and phosphorylated TBK1 using THP-1 (D) or PBMCs (E) transfected with Scr siRNA or VANGL2 siRNA for 24 hours and then infected with VSV (MOI of 0.5) for indicated time. ( F ) Immunoblotting analysis of protein extracts of HEK293T cells treated with CHX (100 μg/ml) for 12 hours, followed by treatment with MG132 (10 μM), Baf A1 (0.2 μM), or both for 6 hours. ( G ) Immunoblotting analysis of HEK293T cells transfected with indicated plasmids for 24 hours, followed by treatment with MG132 (10 μM), 3-MA (10 mM), or Baf A1 (0.2 μM) for 6 hours. ( H ) Immunoblotting analysis using lysates from HEK293T cells transfected with FLAG-VANGL2 or FLAG-EV for 24 hours, followed by treatment with rapamycin (250 nM) for indicated time. ( I ) Immunoblotting analysis using lysates from Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs treated with EBSS for indicated time. ( J ) Immunoblotting analysis of WT, BECLIN -KO, or ATG5 -KO HEK293T cells transfected with indicated plasmids. ( K ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated specific antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; violet, LAMP1 signal; blue, DAPI. Scale bars, 20 μm. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Infection, Labeling, Confocal Microscopy

( A and B ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with indicated Flag-tagged cargo receptors along with HA-TBK1 (A) or HA-VANGL2 (B). ( C ) Cell lysates were harvested after Baf A1 (0.2 μM) treatment (6 hours) for co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with HA-TBK1, FLAG-OPTN, and MYC-VANGL2. ( D ) Immunoblotting analysis of WT and OPTN −/− HEK293T cells transfected with FLAG-TBK1 and MYC-EV or MYC-VANGL2 for 24 hours. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of WT, OPTN -KO, NDP52- KO, p62 -KO, or TOLLIP -KO HEK293T cells transfected with FLAG-TBK1, together with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 for 24 hours. ( F and G ) A549 (F) or BMDMs (G) cells were infected with VSV (MOI of 0.5), and protein lysates were harvested for IP using an anti-OPTN antibody. ( H to J ) Immunoblotting (H) and RT-PCR (I) and (J) analysis of WT and OPTN -KO HEK293T cells transfected with FLAG-EV or FLAG-VANGL2 for 24 hours, followed by treatment with or without VSV (MOI of 0.5) infection for 12 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *** P < 0.001 and **** P < 0.0001 as determined by unpaired Student’s t test.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with indicated Flag-tagged cargo receptors along with HA-TBK1 (A) or HA-VANGL2 (B). ( C ) Cell lysates were harvested after Baf A1 (0.2 μM) treatment (6 hours) for co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with HA-TBK1, FLAG-OPTN, and MYC-VANGL2. ( D ) Immunoblotting analysis of WT and OPTN −/− HEK293T cells transfected with FLAG-TBK1 and MYC-EV or MYC-VANGL2 for 24 hours. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of WT, OPTN -KO, NDP52- KO, p62 -KO, or TOLLIP -KO HEK293T cells transfected with FLAG-TBK1, together with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 for 24 hours. ( F and G ) A549 (F) or BMDMs (G) cells were infected with VSV (MOI of 0.5), and protein lysates were harvested for IP using an anti-OPTN antibody. ( H to J ) Immunoblotting (H) and RT-PCR (I) and (J) analysis of WT and OPTN -KO HEK293T cells transfected with FLAG-EV or FLAG-VANGL2 for 24 hours, followed by treatment with or without VSV (MOI of 0.5) infection for 12 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *** P < 0.001 and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Infection, Reverse Transcription Polymerase Chain Reaction

( A ) HEK293T cells were transfected with FLAG-TBK1 and HA-tagged WT ubiquitin (HA-Ub) or its mutants, together with MYC-EV or MYC-VANGL2 for 24 hours, followed by treatment with Baf A1 (0.2 μM) for 6 hours, followed by co-immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibody. ( B ) IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( C ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with Scr shRNA or other E3 ligase–specific shRNAs for 24 hours, followed by transfected with FLAG-TBK1, together with EV or HA-VANGL2 for 24 hours. ( D ) HEK293T cells were transfected with Scr shRNA or TRIP- specific shRNA for 24 hours, followed by transfected with FLAG-EV or FLAG-VANGL2 for 24 hours; protein lysates were harvested after VSV (MOI of 0.5) infection for 12 hours and Baf A1 (0.2 μM) treatment for 6 hours for IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with MYC-EV or MYC-VANGL2 for 24 hours. ( F ) Co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with HA-K48–linked ubiquitin and MYC-EV or MYC-VANGL2; cell lysates were harvested after Baf A1 (0.2 μM) treatment for 6 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. **** P < 0.0001 as determined by unpaired Student’s t test.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with FLAG-TBK1 and HA-tagged WT ubiquitin (HA-Ub) or its mutants, together with MYC-EV or MYC-VANGL2 for 24 hours, followed by treatment with Baf A1 (0.2 μM) for 6 hours, followed by co-immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibody. ( B ) IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( C ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with Scr shRNA or other E3 ligase–specific shRNAs for 24 hours, followed by transfected with FLAG-TBK1, together with EV or HA-VANGL2 for 24 hours. ( D ) HEK293T cells were transfected with Scr shRNA or TRIP- specific shRNA for 24 hours, followed by transfected with FLAG-EV or FLAG-VANGL2 for 24 hours; protein lysates were harvested after VSV (MOI of 0.5) infection for 12 hours and Baf A1 (0.2 μM) treatment for 6 hours for IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with MYC-EV or MYC-VANGL2 for 24 hours. ( F ) Co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with HA-K48–linked ubiquitin and MYC-EV or MYC-VANGL2; cell lysates were harvested after Baf A1 (0.2 μM) treatment for 6 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Luciferase, Activity Assay, shRNA, Mutagenesis, Co-Immunoprecipitation Assay

( A and B ) Weight (A) and survival (B) of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice ( n = 6 mice per group) after intraperitoneal injection of VSV [1 × 10 8 plaque-forming units (PFU) per mouse]. ( C ) ELISA for IFN-β in serum of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice treated with phosphate-buffered saline (PBS) or infected with VSV (1 × 10 8 PFU per mouse) via intraperitoneal injection for 18 hours. ( D and E ) RT-PCR analysis of Ifnb (D) or VSV-G (E) mRNA in the spleen (left), lungs (center), and liver (right) from mice, as in (C). ( F ) Representative hematoxylin and eosin–stained images of lung sections from mice as in (C). Scale bars, 50 μm. ( G ) Graphical abstract to illustrate how VANGL2 negatively regulates IFN-I signaling upon virus infection. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Weight (A) and survival (B) of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice ( n = 6 mice per group) after intraperitoneal injection of VSV [1 × 10 8 plaque-forming units (PFU) per mouse]. ( C ) ELISA for IFN-β in serum of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice treated with phosphate-buffered saline (PBS) or infected with VSV (1 × 10 8 PFU per mouse) via intraperitoneal injection for 18 hours. ( D and E ) RT-PCR analysis of Ifnb (D) or VSV-G (E) mRNA in the spleen (left), lungs (center), and liver (right) from mice, as in (C). ( F ) Representative hematoxylin and eosin–stained images of lung sections from mice as in (C). Scale bars, 50 μm. ( G ) Graphical abstract to illustrate how VANGL2 negatively regulates IFN-I signaling upon virus infection. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Staining