Journal: Cell Adhesion & Migration

Article Title: DDR1 and DDR2 physical interaction leads to signaling interconnection but with possible distinct functions

doi: 10.1080/19336918.2018.1460012

Figure Lengend Snippet: Primary antibodies used for western blot analysis.

Article Snippet: Antigen From Dilution DDR1 –Santa Cruz Biotechnology (SCB) (Rabbit) 1/1000 –Cell Signaling Technology (CST) (Rabbit) 1/1000 DDR2 –SCB (Goat) 1/1000 –CST (Rabbit) 1/1000 c-Myc –Clone 9E10 1/1000 P-Tyr –Clone 4G10 1/1000 P-Jak2 (Tyr1007-1008) –CST (Rabbit) 1/1000 P-Erk –CST (Rabbit) 1/1000 GAPDH –SCB (Rabbit) 1/2000 Open in a separate window Primary antibodies used for western blot analysis. . Immunoprecipitation 1 mg/500μl of solubilized proteins were incubated with DDR1 or DDR2 antibody (1 μg, SCB; or 1/100 dilution, CST) for 3 h (SCB) or overnight (CST) at 4°C.

Techniques: Western Blot

Journal: Science Advances

Article Title: VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1 β in macrophages

doi: 10.1126/sciadv.aau7426

Figure Lengend Snippet: PEMs and liver tissues were isolated from Vsig4 −/− mice and their C57BL/6 WT littermates. ( A ) Western blot analysis of A20 (each number represents an individual mouse). RAW264.7 cells were transfected with Len-Cont. or Len-Vsig4 . ( B ) Cell extracts were immunoblotted for the indicated protein. ( C ) A20 in RAW264.7 cells was silenced by shRNA , cells were further treated with LPS (2 μg/ml) for an additional 3 hours, and cell extracts were immunoblotted for the indicated protein. ( D ) PEMs were treated with LPS (2 μg/ml) in the presence of NF-κB inhibitor BAY 11-7082 (5 μM), and cell extracts were immunoblotted for the indicated molecules after 6 hours. ( E ) The transcription of Stat3 in RAW264.7 cells was silenced by shRNA , and cell extracts were immunoblotted for A20 and STAT3. LPS-primed RAW264.7 cells were treated with ( F ) the STAT3 inhibitor S3I-201 (100 mM) and ( G ) the JAK2 inhibitor TG101348 (10 μM), and cell extracts were immunoblotted for the indicated molecules after 12 hours. ( H ) ChIP-qPCR analysis of the enrichment of p-STAT3 binding to the potential binding sites in the A20 promoter region in Vsig4 + RAW264.7 cells after 3 hours of LPS (2 μg/ml) administration. ( I ) Analysis of luciferase activity of A20 gene promoter constructs by luciferase reporter assay. Error bars indicate SEM. NS, not significant. * P < 0.05 and ** P < 0.01 (Student’s t test). Data represent one out of three biological replicates, with three technical replicates each at least.

Article Snippet: The expression of GAPDH (#2118, CST, Danvers, MA, USA), actin-β (#3700, CST), Akt (#2920, CST), p-Akt (p-Thy 308 , #4056; p-Ser 473 , #4051, CST), STAT3 (#9139, CST), p-STAT3 (p-Tyr 705 , #9145, CST), Stat6 (#5397, CST), p-Stat6 (p-Tyr 641 , #56554, CST), p-p65 (#3033, CST), Jak1 (#3344, CST), p-Jak1 (#3331, CST), Jak2 (#3230, CST), p-Jak2 (#3771, CST), ASC (#sc-376916, Santa Cruz Biotechnology), IL-18 (#ab71495, Abcam), IL-1β (#ab9722, Abcam), caspase-1 (AG-20B-0042, Adipogen, San Diego, CA, USA), and NLRP3 (#AG-20B-0014-C100, Adipogen) in macrophages, liver tissues, and spinal cords were measured by Western blot, as described previously ( ).

Techniques: Isolation, Western Blot, Transfection, shRNA, ChIP-qPCR, Binding Assay, Luciferase, Activity Assay, Construct, Reporter Assay

Journal: Science Advances

Article Title: VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1 β in macrophages

doi: 10.1126/sciadv.aau7426

Figure Lengend Snippet: PEMs from C57BL/6 WT mice were isolated. ( A ) Immunofluoresence double-staining analysis of the coexpression of VSIG4 and MS4A6D. Arrows indicate positive cells, and blue indicates nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 20 μm. ( B ) PEMs were treated with the VG11 mAbs (100 μg/ml) at indicated times, and cell lysates were used for IP with anti-VSIG4 and blotting with anti-MS4A6D. ( C ) Ms4a6d –HA (hemagglutinin) and Vsig4 -Flag were coexpressed in RAW264.7 cells, cell lysates were collected, and VSIG4/MS4A6D interactions were analyzed by Co-IP experiments. ( D ) Ms4a6d -HA and Vsig4 -Flag were cotransfected in RAW264.7 cells, cells were further triggered by VG11 mAbs (100 μg/ml), and MS4A6D and JAK2 interactions were analyzed by Co-IP experiments. ( E ) PEMs were isolated and further treated with VG11 mAbs (100 μg/ml) for 12 hours, and cell extracts were immunoblotted for p-STAT3/STAT3 and A20. ( F ) PEMs were isolated from Ms4a6d −/− and WT mice, cells were further treated with LPS (2 μg/ml) for 0 or 3 hours, the transcription of Nlrp3 and Il-1 β was analyzed by qRT-PCR. ( G ) Ms4a6d in Vsig4 + RAW264.7 cells was silenced by shRNA, and cell extracts were immunoblotted for the indicated molecules. ( H ) Overexpression of Ms4a6d in Vsig4 + RAW264.7 cells, cells were further treated with LPS (2 μg/ml) for 6 h, and cell extracts were immunoblotted for p-STAT3/STAT3 and A20. ( I ) Cell extracts were immunoblotted for A20 from Ms4a6d −/− PEMs that were transfected with different Ms4a6d deletion constructs. ( J ) Vsig4 + RAW264.7 cells were treated with VG11 mAbs (100 μg/ml), MS4A6D protein was pulled down by IP experiments, and Ser phosphorylation (p-Ser) was analyzed by Western blot. ( K ) Ms4a6d −/− PEMs were transfected with different Ms4a6d mutations, and cell extracts were immunoblotted for A20. Error bars indicate SEM. * P < 0.05 and ** P < 0.01 (Student’s t test). Data represent one out of three biological replicates, with three technical replicates each at least.

Article Snippet: The expression of GAPDH (#2118, CST, Danvers, MA, USA), actin-β (#3700, CST), Akt (#2920, CST), p-Akt (p-Thy 308 , #4056; p-Ser 473 , #4051, CST), STAT3 (#9139, CST), p-STAT3 (p-Tyr 705 , #9145, CST), Stat6 (#5397, CST), p-Stat6 (p-Tyr 641 , #56554, CST), p-p65 (#3033, CST), Jak1 (#3344, CST), p-Jak1 (#3331, CST), Jak2 (#3230, CST), p-Jak2 (#3771, CST), ASC (#sc-376916, Santa Cruz Biotechnology), IL-18 (#ab71495, Abcam), IL-1β (#ab9722, Abcam), caspase-1 (AG-20B-0042, Adipogen, San Diego, CA, USA), and NLRP3 (#AG-20B-0014-C100, Adipogen) in macrophages, liver tissues, and spinal cords were measured by Western blot, as described previously ( ).

Techniques: Isolation, Double Staining, Staining, Co-Immunoprecipitation Assay, Quantitative RT-PCR, shRNA, Over Expression, Transfection, Construct, Phospho-proteomics, Western Blot

Journal: Science Advances

Article Title: VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1 β in macrophages

doi: 10.1126/sciadv.aau7426

Figure Lengend Snippet: PEMs and liver tissues were isolated from Vsig4 −/− mice and their C57BL/6 WT littermates. ( A ) Western blot analysis of A20 (each number represents an individual mouse). RAW264.7 cells were transfected with Len-Cont. or Len-Vsig4 . ( B ) Cell extracts were immunoblotted for the indicated protein. ( C ) A20 in RAW264.7 cells was silenced by shRNA , cells were further treated with LPS (2 μg/ml) for an additional 3 hours, and cell extracts were immunoblotted for the indicated protein. ( D ) PEMs were treated with LPS (2 μg/ml) in the presence of NF-κB inhibitor BAY 11-7082 (5 μM), and cell extracts were immunoblotted for the indicated molecules after 6 hours. ( E ) The transcription of Stat3 in RAW264.7 cells was silenced by shRNA , and cell extracts were immunoblotted for A20 and STAT3. LPS-primed RAW264.7 cells were treated with ( F ) the STAT3 inhibitor S3I-201 (100 mM) and ( G ) the JAK2 inhibitor TG101348 (10 μM), and cell extracts were immunoblotted for the indicated molecules after 12 hours. ( H ) ChIP-qPCR analysis of the enrichment of p-STAT3 binding to the potential binding sites in the A20 promoter region in Vsig4 + RAW264.7 cells after 3 hours of LPS (2 μg/ml) administration. ( I ) Analysis of luciferase activity of A20 gene promoter constructs by luciferase reporter assay. Error bars indicate SEM. NS, not significant. * P < 0.05 and ** P < 0.01 (Student’s t test). Data represent one out of three biological replicates, with three technical replicates each at least.

Article Snippet: The expression of GAPDH (#2118, CST, Danvers, MA, USA), actin-β (#3700, CST), Akt (#2920, CST), p-Akt (p-Thy 308 , #4056; p-Ser 473 , #4051, CST), STAT3 (#9139, CST), p-STAT3 (p-Tyr 705 , #9145, CST), Stat6 (#5397, CST), p-Stat6 (p-Tyr 641 , #56554, CST), p-p65 (#3033, CST), Jak1 (#3344, CST), p-Jak1 (#3331, CST), Jak2 (#3230, CST), p-Jak2 (#3771, CST), ASC (#sc-376916, Santa Cruz Biotechnology), IL-18 (#ab71495, Abcam), IL-1β (#ab9722, Abcam), caspase-1 (AG-20B-0042, Adipogen, San Diego, CA, USA), and NLRP3 (#AG-20B-0014-C100, Adipogen) in macrophages, liver tissues, and spinal cords were measured by Western blot, as described previously ( ).

Techniques: Isolation, Western Blot, Transfection, shRNA, ChIP-qPCR, Binding Assay, Luciferase, Activity Assay, Construct, Reporter Assay

Journal: Science Advances

Article Title: VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1 β in macrophages

doi: 10.1126/sciadv.aau7426

Figure Lengend Snippet: PEMs from C57BL/6 WT mice were isolated. ( A ) Immunofluoresence double-staining analysis of the coexpression of VSIG4 and MS4A6D. Arrows indicate positive cells, and blue indicates nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 20 μm. ( B ) PEMs were treated with the VG11 mAbs (100 μg/ml) at indicated times, and cell lysates were used for IP with anti-VSIG4 and blotting with anti-MS4A6D. ( C ) Ms4a6d –HA (hemagglutinin) and Vsig4 -Flag were coexpressed in RAW264.7 cells, cell lysates were collected, and VSIG4/MS4A6D interactions were analyzed by Co-IP experiments. ( D ) Ms4a6d -HA and Vsig4 -Flag were cotransfected in RAW264.7 cells, cells were further triggered by VG11 mAbs (100 μg/ml), and MS4A6D and JAK2 interactions were analyzed by Co-IP experiments. ( E ) PEMs were isolated and further treated with VG11 mAbs (100 μg/ml) for 12 hours, and cell extracts were immunoblotted for p-STAT3/STAT3 and A20. ( F ) PEMs were isolated from Ms4a6d −/− and WT mice, cells were further treated with LPS (2 μg/ml) for 0 or 3 hours, the transcription of Nlrp3 and Il-1 β was analyzed by qRT-PCR. ( G ) Ms4a6d in Vsig4 + RAW264.7 cells was silenced by shRNA, and cell extracts were immunoblotted for the indicated molecules. ( H ) Overexpression of Ms4a6d in Vsig4 + RAW264.7 cells, cells were further treated with LPS (2 μg/ml) for 6 h, and cell extracts were immunoblotted for p-STAT3/STAT3 and A20. ( I ) Cell extracts were immunoblotted for A20 from Ms4a6d −/− PEMs that were transfected with different Ms4a6d deletion constructs. ( J ) Vsig4 + RAW264.7 cells were treated with VG11 mAbs (100 μg/ml), MS4A6D protein was pulled down by IP experiments, and Ser phosphorylation (p-Ser) was analyzed by Western blot. ( K ) Ms4a6d −/− PEMs were transfected with different Ms4a6d mutations, and cell extracts were immunoblotted for A20. Error bars indicate SEM. * P < 0.05 and ** P < 0.01 (Student’s t test). Data represent one out of three biological replicates, with three technical replicates each at least.

Article Snippet: The expression of GAPDH (#2118, CST, Danvers, MA, USA), actin-β (#3700, CST), Akt (#2920, CST), p-Akt (p-Thy 308 , #4056; p-Ser 473 , #4051, CST), STAT3 (#9139, CST), p-STAT3 (p-Tyr 705 , #9145, CST), Stat6 (#5397, CST), p-Stat6 (p-Tyr 641 , #56554, CST), p-p65 (#3033, CST), Jak1 (#3344, CST), p-Jak1 (#3331, CST), Jak2 (#3230, CST), p-Jak2 (#3771, CST), ASC (#sc-376916, Santa Cruz Biotechnology), IL-18 (#ab71495, Abcam), IL-1β (#ab9722, Abcam), caspase-1 (AG-20B-0042, Adipogen, San Diego, CA, USA), and NLRP3 (#AG-20B-0014-C100, Adipogen) in macrophages, liver tissues, and spinal cords were measured by Western blot, as described previously ( ).

Techniques: Isolation, Double Staining, Staining, Co-Immunoprecipitation Assay, Quantitative RT-PCR, shRNA, Over Expression, Transfection, Construct, Phospho-proteomics, Western Blot