Structured Review

Upstate Biotechnology Inc stat1
DN forms of CaMKII inhibit <t>Stat1</t> S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Stat1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/stat1/product/Upstate Biotechnology Inc
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
stat1 - by Bioz Stars, 2020-11
92/100 stars

Images

1) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

2) Product Images from "Etk, a Btk Family Tyrosine Kinase, Mediates Cellular Transformation by Linking Src to STAT3 Activation"

Article Title: Etk, a Btk Family Tyrosine Kinase, Mediates Cellular Transformation by Linking Src to STAT3 Activation

Journal: Molecular and Cellular Biology

doi:

Etk mediates the activation of STAT3 by v-Src. (A) The kinase-defective mutant of Etk (EtkKQ) functions as a dominant-negative mutant. The upper blot shows the expression levels of Etk and EtkKQ in parental WB cell and four stable cell lines as demonstrated by immunoblotting with anti-Etk antibody. The lower blot shows Etk kinase activities in WB and its stable lines. EtkKQ-M is a mixture of all four stable clones. Lysates of cells were immunoprecipitated with anti-Etk antibody, followed by in vitro kinase assays. The autophosphorylated Etk is shown. (B) Introducing v-Src into WB and its derivatives by recombinant retrovirus. WB and its derivatives were infected by retrovirus containing v-Src and the puromycin resistance gene. Lysates from equal numbers of puromycin resistant cells from each infection were used for Western blotting with antibody specific to v-Src. (C) EtkKQ blocks STAT3 DNA binding activity induced by v-Src. Nuclear extracts from WB and its derivatives as indicated were subjected to EMSA using a 32 P-labeled hSIE probe. A 133-fold molar excess of cold hSIE or a nonspecific oligonucleotide (NS) was used as the competitor, and supershifting (STAT3* indicates supershifted complex) was performed with anti-STAT3 antibody. The anti-STAT1 antibody was included as a control. (D) EtkKQ inhibits v-Src-induced tyrosine phosphorylation of STAT3. Lysates from cells as indicated were used for Western blotting with an antibody specific to STAT3 phosphorylated at position 705 (phospho-STAT) (upper blot) or the anti-STAT3 antibody (lower blot). (E) EtkKQ does not generally block tyrosine phosphorylation on v-Src targets. Lysates from cells as indicated were used for Western blotting with antiphosphotyrosine antibody (anti-pY) or antitubulin antibody.
Figure Legend Snippet: Etk mediates the activation of STAT3 by v-Src. (A) The kinase-defective mutant of Etk (EtkKQ) functions as a dominant-negative mutant. The upper blot shows the expression levels of Etk and EtkKQ in parental WB cell and four stable cell lines as demonstrated by immunoblotting with anti-Etk antibody. The lower blot shows Etk kinase activities in WB and its stable lines. EtkKQ-M is a mixture of all four stable clones. Lysates of cells were immunoprecipitated with anti-Etk antibody, followed by in vitro kinase assays. The autophosphorylated Etk is shown. (B) Introducing v-Src into WB and its derivatives by recombinant retrovirus. WB and its derivatives were infected by retrovirus containing v-Src and the puromycin resistance gene. Lysates from equal numbers of puromycin resistant cells from each infection were used for Western blotting with antibody specific to v-Src. (C) EtkKQ blocks STAT3 DNA binding activity induced by v-Src. Nuclear extracts from WB and its derivatives as indicated were subjected to EMSA using a 32 P-labeled hSIE probe. A 133-fold molar excess of cold hSIE or a nonspecific oligonucleotide (NS) was used as the competitor, and supershifting (STAT3* indicates supershifted complex) was performed with anti-STAT3 antibody. The anti-STAT1 antibody was included as a control. (D) EtkKQ inhibits v-Src-induced tyrosine phosphorylation of STAT3. Lysates from cells as indicated were used for Western blotting with an antibody specific to STAT3 phosphorylated at position 705 (phospho-STAT) (upper blot) or the anti-STAT3 antibody (lower blot). (E) EtkKQ does not generally block tyrosine phosphorylation on v-Src targets. Lysates from cells as indicated were used for Western blotting with antiphosphotyrosine antibody (anti-pY) or antitubulin antibody.

Techniques Used: Activation Assay, Mutagenesis, Dominant Negative Mutation, Expressing, Western Blot, Stable Transfection, Clone Assay, Immunoprecipitation, In Vitro, Recombinant, Infection, Binding Assay, Activity Assay, Labeling, Blocking Assay

3) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

4) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

5) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

6) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

7) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

8) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

9) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

10) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

11) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

12) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

13) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

14) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

15) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

16) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

17) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

18) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

19) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

20) Product Images from "Global changes in STAT target selection and transcription regulation upon interferon treatments"

Article Title: Global changes in STAT target selection and transcription regulation upon interferon treatments

Journal: Genes & Development

doi: 10.1101/gad.1371305

Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and IFN-α–STAT2 ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .
Figure Legend Snippet: Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and IFN-α–STAT2 ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .

Techniques Used: Chromatin Immunoprecipitation, Expressing, Microarray, Binding Assay

IFN stimulations activate the STATs and induce their binding to correct DNA target sequences. ( A ) Western blotting of STAT1 and STAT2 immunoprecipitations from nuclear extracts detected the increase in nuclear localization and phosphorylation of STAT1 and STAT2. ( B ) PCR analysis of STAT1 and STAT2 ChIP DNA confirmed the IFN-induced enriched presence of the STAT-binding sites in the IRF1 and OAS1 promoters. Positive and negative controls used genomic DNA and no template, respectively.
Figure Legend Snippet: IFN stimulations activate the STATs and induce their binding to correct DNA target sequences. ( A ) Western blotting of STAT1 and STAT2 immunoprecipitations from nuclear extracts detected the increase in nuclear localization and phosphorylation of STAT1 and STAT2. ( B ) PCR analysis of STAT1 and STAT2 ChIP DNA confirmed the IFN-induced enriched presence of the STAT-binding sites in the IRF1 and OAS1 promoters. Positive and negative controls used genomic DNA and no template, respectively.

Techniques Used: Binding Assay, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation

Schematic of IFN-induced STAT1-binding site selection. ( A ) IFN-γ and IFN-α both induce STAT1 homodimers, which bind DNA (Gene A); however, we also observed IFN-α-induced, STAT2-independent binding of STAT1 to sites not occupied by IFN-γ-induced STAT1 (Gene C). ( B ) Changes in binding site accessibility and/or the presence of a cooperating cofactor may account for the binding of STAT1 to new sites upon IFN-α treatment. Also, the binding of STAT1 to alternate dimerization partners (such as STAT2) reduces the relative quantity of STAT1 homodimers; thus, a loss of STAT1 binding to some IFN-γ-induced sites occurs.
Figure Legend Snippet: Schematic of IFN-induced STAT1-binding site selection. ( A ) IFN-γ and IFN-α both induce STAT1 homodimers, which bind DNA (Gene A); however, we also observed IFN-α-induced, STAT2-independent binding of STAT1 to sites not occupied by IFN-γ-induced STAT1 (Gene C). ( B ) Changes in binding site accessibility and/or the presence of a cooperating cofactor may account for the binding of STAT1 to new sites upon IFN-α treatment. Also, the binding of STAT1 to alternate dimerization partners (such as STAT2) reduces the relative quantity of STAT1 homodimers; thus, a loss of STAT1 binding to some IFN-γ-induced sites occurs.

Techniques Used: Binding Assay, Selection

21) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

22) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

23) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

24) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

25) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

26) Product Images from "IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells"

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087668

IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p
Figure Legend Snippet: IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p

Techniques Used: Expressing, Fluorescence

27) Product Images from "JAK1-STAT1-STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts"

Article Title: JAK1-STAT1-STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200703184

JAK1/STAT1/STAT3 were activated during cardiotoxin-induced muscle regeneration. (A) After the injection of cardiotoxin (CTX), TA muscles were isolated at different time points as indicated. WCEs from TA muscles were prepared, and 200 μg WCE was subjected to immunoblotting. (B) WCEs from control and regenerating TA muscles were separately subjected to immunoprecipitation (IP) with either the anti-JAK1 antibody (top two panels) or the anti-STAT1 antibody (bottom two panels). The immunoprecipitated JAK1 was subjected to both protein kinase assays (top) and immunoblotting (second panel), whereas the immunoprecipitated STAT1 was only subjected to immunoblotting (third and fourth panels). (C) A schematic on injury-induced muscle regeneration. Key molecules/pathways acting at different stages of regeneration are indicated.
Figure Legend Snippet: JAK1/STAT1/STAT3 were activated during cardiotoxin-induced muscle regeneration. (A) After the injection of cardiotoxin (CTX), TA muscles were isolated at different time points as indicated. WCEs from TA muscles were prepared, and 200 μg WCE was subjected to immunoblotting. (B) WCEs from control and regenerating TA muscles were separately subjected to immunoprecipitation (IP) with either the anti-JAK1 antibody (top two panels) or the anti-STAT1 antibody (bottom two panels). The immunoprecipitated JAK1 was subjected to both protein kinase assays (top) and immunoblotting (second panel), whereas the immunoprecipitated STAT1 was only subjected to immunoblotting (third and fourth panels). (C) A schematic on injury-induced muscle regeneration. Key molecules/pathways acting at different stages of regeneration are indicated.

Techniques Used: Injection, Isolation, Immunoprecipitation

STAT1 mediates the antidifferentiation effect of JAK1. (A) C2C12 cells were transfected with JAK1-siRNA together with various forms of JAK1 cDNA. After 24 h in DM, cells were harvested, and WCEs were subjected to immunoblotting. R, siRNA resistant; KR, kinase-dead mutant of JAK1 with Lys896 replaced by Arg. (B) C2C12 cells were transfected with various siRNAs as indicated. WCEs were prepared from cells grown in DM for 12 h and subjected to immunoblotting. ns, nonspecific band. (C) 100 μg WCE prepared from primary myoblasts harvested at different times was subjected to immunoblotting. (D) Freshly isolated primary myoblasts were transfected with either GFP-siRNA or STAT1-siRNA. 24 h after transfection, cells were fixed and subjected to immunostaining for MHC (top). The nuclei of the cells were counterstained with DAPI (bottom). The percentage of MHC-positive cells was calculated based on cells from five randomly chosen fields. Error bars represent SD. (E) C2C12 cells were cotransfected with various siRNAs and cDNA expression vectors as indicated. After 36 h in DM, cells were harvested, and WCEs were subjected to immunoblotting. Bar, 100 μm.
Figure Legend Snippet: STAT1 mediates the antidifferentiation effect of JAK1. (A) C2C12 cells were transfected with JAK1-siRNA together with various forms of JAK1 cDNA. After 24 h in DM, cells were harvested, and WCEs were subjected to immunoblotting. R, siRNA resistant; KR, kinase-dead mutant of JAK1 with Lys896 replaced by Arg. (B) C2C12 cells were transfected with various siRNAs as indicated. WCEs were prepared from cells grown in DM for 12 h and subjected to immunoblotting. ns, nonspecific band. (C) 100 μg WCE prepared from primary myoblasts harvested at different times was subjected to immunoblotting. (D) Freshly isolated primary myoblasts were transfected with either GFP-siRNA or STAT1-siRNA. 24 h after transfection, cells were fixed and subjected to immunostaining for MHC (top). The nuclei of the cells were counterstained with DAPI (bottom). The percentage of MHC-positive cells was calculated based on cells from five randomly chosen fields. Error bars represent SD. (E) C2C12 cells were cotransfected with various siRNAs and cDNA expression vectors as indicated. After 36 h in DM, cells were harvested, and WCEs were subjected to immunoblotting. Bar, 100 μm.

Techniques Used: Transfection, Mutagenesis, Isolation, Immunostaining, Expressing

LIF stimulates proliferation and represses differentiation via JAK1–STAT1–STAT3. (A) C2C12 cells were treated with either vehicle (i.e., PBS) or LIF. At different time points, cells in 96-well plates (five wells/time point/sample) were subjected to WST-1 assays. The absorbance at 480 nm was plotted against time. The results are presented as mean ± SD (error bars). (B) Cells were treated with LIF for 15 min before harvest. 20 μg WCE was subjected to EMSA. NS, nonspecific. (C) Cells were treated with LIF for various times as indicated. (D) Cells were first transfected with either GFP-siRNA or JAK1-siRNA followed by LIF treatment for 15 min. (E) C2C12 cells were treated with LIF for various times. (F) Cells were first transfected with various siRNAs as indicated followed by LIF treatment for 12 h in DM. WCEs from C–F were subjected to immunoblotting. (G) Cells in 96-well plates were transfected with siRNAs followed by LIF treatment for various times before being subjected to WST-1 assays. The results are plotted and presented the same way as in A. The plus and minus signs indicate that the reagents listed on the left were present and absent, respectively. The white line indicates that intervening lanes have been spliced out.
Figure Legend Snippet: LIF stimulates proliferation and represses differentiation via JAK1–STAT1–STAT3. (A) C2C12 cells were treated with either vehicle (i.e., PBS) or LIF. At different time points, cells in 96-well plates (five wells/time point/sample) were subjected to WST-1 assays. The absorbance at 480 nm was plotted against time. The results are presented as mean ± SD (error bars). (B) Cells were treated with LIF for 15 min before harvest. 20 μg WCE was subjected to EMSA. NS, nonspecific. (C) Cells were treated with LIF for various times as indicated. (D) Cells were first transfected with either GFP-siRNA or JAK1-siRNA followed by LIF treatment for 15 min. (E) C2C12 cells were treated with LIF for various times. (F) Cells were first transfected with various siRNAs as indicated followed by LIF treatment for 12 h in DM. WCEs from C–F were subjected to immunoblotting. (G) Cells in 96-well plates were transfected with siRNAs followed by LIF treatment for various times before being subjected to WST-1 assays. The results are plotted and presented the same way as in A. The plus and minus signs indicate that the reagents listed on the left were present and absent, respectively. The white line indicates that intervening lanes have been spliced out.

Techniques Used: Transfection

28) Product Images from "Herpes Simplex Virus 1 Gene Products Occlude the Interferon Signaling Pathway at Multiple Sites"

Article Title: Herpes Simplex Virus 1 Gene Products Occlude the Interferon Signaling Pathway at Multiple Sites

Journal: Journal of Virology

doi: 10.1128/JVI.78.8.4185-4196.2004

Effect of HSV-1 on members of the Stat family of proteins. (A) Replicate HeLa cells were mock infected or inoculated with 10 PFU of HSV-1(F) per cell as described in Materials and Methods. After 7 h of incubation, the cells were either not treated or treated with 500 U of IFN-γ/ml for 1 h or with 5,000 U of IFN-α/ml for 15 min. The cells were harvested and fractionated, and nuclear and cytoplasmic fractions (200 μg/lane) were electrophoretically separated on denaturing polyacrylamide gels, transferred to nitrocellulose membranes, and reacted with antibodies to Stat1, Stat2, Stat3, Stat5, or Stat6 (Transduction Laboratories) and then with secondary antibodies conjugated to AP. AP-conjugated antibodies were detected by using a colorimetric reaction (Bio-Rad). (B) Replicate HeLa cells were mock infected or exposed to 5 PFU of wild-type virus per cell or 5 PFU of mutant viruses lacking U L 41, γ 1 34.5, α47, U S 9, U S 3, U L 13, α22, or α0 genes per cell. The cells were harvested at 18 h after infection and processed as described in Materials and Methods. Whole-cell lysates were subjected to electrophoresis in denaturing gels (200 μg/lane) and reacted with antibodies as described above. Bound peroxidase-conjugated antibodies were detected with the aid of enhanced chemiluminescence.
Figure Legend Snippet: Effect of HSV-1 on members of the Stat family of proteins. (A) Replicate HeLa cells were mock infected or inoculated with 10 PFU of HSV-1(F) per cell as described in Materials and Methods. After 7 h of incubation, the cells were either not treated or treated with 500 U of IFN-γ/ml for 1 h or with 5,000 U of IFN-α/ml for 15 min. The cells were harvested and fractionated, and nuclear and cytoplasmic fractions (200 μg/lane) were electrophoretically separated on denaturing polyacrylamide gels, transferred to nitrocellulose membranes, and reacted with antibodies to Stat1, Stat2, Stat3, Stat5, or Stat6 (Transduction Laboratories) and then with secondary antibodies conjugated to AP. AP-conjugated antibodies were detected by using a colorimetric reaction (Bio-Rad). (B) Replicate HeLa cells were mock infected or exposed to 5 PFU of wild-type virus per cell or 5 PFU of mutant viruses lacking U L 41, γ 1 34.5, α47, U S 9, U S 3, U L 13, α22, or α0 genes per cell. The cells were harvested at 18 h after infection and processed as described in Materials and Methods. Whole-cell lysates were subjected to electrophoresis in denaturing gels (200 μg/lane) and reacted with antibodies as described above. Bound peroxidase-conjugated antibodies were detected with the aid of enhanced chemiluminescence.

Techniques Used: Infection, Incubation, Transduction, Mutagenesis, Electrophoresis

HSV-1 blocks the formation of DNA protein complexes induced by IFN. Replicate HEp2 cells (A and B) or HeLa cells (C and D) were mock infected or exposed to10 PFU of HSV-1(F) per cell as described in Materials and Methods After 7 h of incubation, the cells were treated (+) or not treated (−) with 500 U of IFN-γ/ml for 1 h (A and B) or 5,000 U of IFN-α/ml for 15 min (C and D). Cells were harvested, fractionated, and processed as described in Materials and Methods. (A and C) EMSAs. Nuclear (lanes 1 to 4) or cytoplasmic fractions (40 μg/reaction) were reacted with either labeled M67 probe (A) or ISRE probe (C) in total volumes of 30 μl per lane. Complexes containing Stat1 (circles, A) or ISGF3 (C) are identified by open circles to the left of the bands. Bands containing virus-induced proteins are identified with a “v” placed to the left of the bands. Although not specifically identified as such here, HSV-1 induces proteins capable of binding DNA, and this could account for the virus-specific bands. (B and D) Supershift assays. To the reaction mixtures of untreated and IFN-treated cells described above were added 1 μl of Stat1 (lane 3), Stat2 (lane 4), or Stat3 (lane 5) antibody (A) or 1 μl of Stat1 (lane 3), Stat2 (lane 4), ISGF3 p48 (lane 5), or Stat6 (lane 6) antibody (D). The reaction mixtures were incubated for an additional 30 min and then subjected to electrophoresis in denaturing gels. The bands supershifted by antibodies are marked by an open triangle to the left of the band.
Figure Legend Snippet: HSV-1 blocks the formation of DNA protein complexes induced by IFN. Replicate HEp2 cells (A and B) or HeLa cells (C and D) were mock infected or exposed to10 PFU of HSV-1(F) per cell as described in Materials and Methods After 7 h of incubation, the cells were treated (+) or not treated (−) with 500 U of IFN-γ/ml for 1 h (A and B) or 5,000 U of IFN-α/ml for 15 min (C and D). Cells were harvested, fractionated, and processed as described in Materials and Methods. (A and C) EMSAs. Nuclear (lanes 1 to 4) or cytoplasmic fractions (40 μg/reaction) were reacted with either labeled M67 probe (A) or ISRE probe (C) in total volumes of 30 μl per lane. Complexes containing Stat1 (circles, A) or ISGF3 (C) are identified by open circles to the left of the bands. Bands containing virus-induced proteins are identified with a “v” placed to the left of the bands. Although not specifically identified as such here, HSV-1 induces proteins capable of binding DNA, and this could account for the virus-specific bands. (B and D) Supershift assays. To the reaction mixtures of untreated and IFN-treated cells described above were added 1 μl of Stat1 (lane 3), Stat2 (lane 4), or Stat3 (lane 5) antibody (A) or 1 μl of Stat1 (lane 3), Stat2 (lane 4), ISGF3 p48 (lane 5), or Stat6 (lane 6) antibody (D). The reaction mixtures were incubated for an additional 30 min and then subjected to electrophoresis in denaturing gels. The bands supershifted by antibodies are marked by an open triangle to the left of the band.

Techniques Used: Infection, Incubation, Labeling, Binding Assay, Electrophoresis

Effect of HSV-1 on the accumulation of Stat1 and Stat2 protein levels. Replicate HeLa cell cultures were either mock infected or exposed to 10 PFU of HSV-1(F) per cell and then harvested at 2, 4, 8, 12, or 16 h after infection, processed, subjected to electrophoresis in denaturing gels (200 μg of protein per lane), transferred to a nitrocellulose sheet, and reacted with anti-Stat1 (A) or Stat2 (B) antibodies and then with secondary antibodies conjugated to peroxidase. Bound antibodies were detected by using enhanced chemiluminescence and quantified with the aid of the Storm 860 PhosphorImager. The results were normalized to the Stat protein levels in the uninfected cell samples.
Figure Legend Snippet: Effect of HSV-1 on the accumulation of Stat1 and Stat2 protein levels. Replicate HeLa cell cultures were either mock infected or exposed to 10 PFU of HSV-1(F) per cell and then harvested at 2, 4, 8, 12, or 16 h after infection, processed, subjected to electrophoresis in denaturing gels (200 μg of protein per lane), transferred to a nitrocellulose sheet, and reacted with anti-Stat1 (A) or Stat2 (B) antibodies and then with secondary antibodies conjugated to peroxidase. Bound antibodies were detected by using enhanced chemiluminescence and quantified with the aid of the Storm 860 PhosphorImager. The results were normalized to the Stat protein levels in the uninfected cell samples.

Techniques Used: Infection, Electrophoresis

HSV-1 infection can inhibit tyrosine phosphorylation but not serine phosphorylation of Stat1 in HeLa cells. Replicate HeLa cell cultures were mock infected or exposed to 10 PFU of HSV-1(F) per cell. At 7 h after mock infection or infection the cells were mock treated or exposed to 500 U of IFN-γ/ml for 1 h or 5,000 U of IFN-α/ml for 15 min. At the conclusion of the treatment the cells were harvested and fractionated, and nuclear and cytoplasmic fractions were subjected to electrophoresis in denaturing gels (200 μg/lane). The electrophoretically separated proteins were transferred to a nitrocellulose sheet and reacted with primary antibodies to phospho-epitope-specific polyclonal antibodies tyrosine 701 or serine 727 of Stat1 (Upstate Biotechnology) and then with a secondary antibody conjugated to peroxidase. Bound peroxidase-conjugated antibodies were detected by using enhanced chemiluminescence.
Figure Legend Snippet: HSV-1 infection can inhibit tyrosine phosphorylation but not serine phosphorylation of Stat1 in HeLa cells. Replicate HeLa cell cultures were mock infected or exposed to 10 PFU of HSV-1(F) per cell. At 7 h after mock infection or infection the cells were mock treated or exposed to 500 U of IFN-γ/ml for 1 h or 5,000 U of IFN-α/ml for 15 min. At the conclusion of the treatment the cells were harvested and fractionated, and nuclear and cytoplasmic fractions were subjected to electrophoresis in denaturing gels (200 μg/lane). The electrophoretically separated proteins were transferred to a nitrocellulose sheet and reacted with primary antibodies to phospho-epitope-specific polyclonal antibodies tyrosine 701 or serine 727 of Stat1 (Upstate Biotechnology) and then with a secondary antibody conjugated to peroxidase. Bound peroxidase-conjugated antibodies were detected by using enhanced chemiluminescence.

Techniques Used: Infection, Electrophoresis

29) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

30) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

31) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

32) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

33) Product Images from "IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells"

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087668

IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p
Figure Legend Snippet: IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p

Techniques Used: Expressing, Fluorescence

34) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

35) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

36) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

37) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

38) Product Images from "Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?"

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.052159099

DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure Legend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques Used: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.
Figure Legend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

Techniques Used: Activation Assay, Western Blot, Northern Blot

CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.
Figure Legend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

Techniques Used: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

39) Product Images from "IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells"

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087668

IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p
Figure Legend Snippet: IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p

Techniques Used: Expressing, Fluorescence

40) Product Images from "Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation"

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11476-11483.2002

Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
Figure Legend Snippet: Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).

Techniques Used: Transfection, Plasmid Preparation, Expressing, SDS Page, Molecular Weight, Immunoprecipitation, Filtration, Chromatography

Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.
Figure Legend Snippet: Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2 − ). Arrows indicate Nipah V protein-expressing cells.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Blocking Assay, Translocation Assay, Fluorescence

Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
Figure Legend Snippet: Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Modification, FLAG-tag

Related Articles

Transfection:

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?
Article Snippet: .. Furthermore, to demonstrate that the CaMKII mutants can inhibit IFN-γ-induced transcription of an endogenous gene, U3A cells were transiently transfected with Stat1 and the WT or the CaMKII DN mutant followed by reverse transcription–PCR analyses of total RNA. ..

Mutagenesis:

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?
Article Snippet: .. Furthermore, to demonstrate that the CaMKII mutants can inhibit IFN-γ-induced transcription of an endogenous gene, U3A cells were transiently transfected with Stat1 and the WT or the CaMKII DN mutant followed by reverse transcription–PCR analyses of total RNA. ..

Functional Assay:

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?
Article Snippet: .. Altogether, these results demonstrate that there is a functional enzyme–substrate interaction between Stat1 and CaMKII. .. To demonstrate further the specific role of CaMKII in Stat1 S727 phosphorylation and transcription activation, DN mutants of CaMKII were generated: CaMKIIγ-A (see Fig. D ), and the ATP-binding-deficient kinase with a K43M mutation (DN).

Activation Assay:

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?
Article Snippet: .. Our results suggest that in NIH 3T3 cells, the Ca2+ flux caused by IFN-γ activates the multifunctional CaMKII, which in turn phosphorylates Stat1 on S727 for maximum transcription activation. .. The specific interaction between Stat1 and CaMKII also suggests the possibility that CaMKII may phosphorylate sites in chromatin to participate in chromatin remodeling near the Stat1 binding sites, which may occur in addition to the acetylation of histones by CBP/p300, also recruited by Stat1 ( , ).

Incubation:

Article Title: Comparison of the effects of food versus protein restriction on selected nutritional and inflammatory markers in rats
Article Snippet: .. Blots were washed and incubated overnight at 4°C with antiphospho-STAT1, anti-STAT1, antiphospho-STAT3, and anti-STAT3 antibodies (Upstate Biotechnology, Lake Placid, NY), or with antiactive MAP kinases (ERK1/ERK2) and anti-MAP kinases antibodies (Promega, Madison, WI), or with phosphor-p38 and anti-p38 kinase antibodies (New England Biolabs, Beverly, MA). .. Goat-rabbit immunoglobulin G (Jackson ImmunoResearch Biotech, Piscataway, NJ) or mouse immunoglobulin G (Upstate Biotechnology, Lake Placid, NY) was used as second antibodies.

other:

Article Title: Etk, a Btk Family Tyrosine Kinase, Mediates Cellular Transformation by Linking Src to STAT3 Activation
Article Snippet: Antibodies to JAK2, v-Src, c-Src, STAT1, and STAT3 were from Upstate Biotechnology.

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?
Article Snippet: Phosphorylation of S727 of Stat1 was inhibited substantially by BAPTA, whereas phosphorylation of Y701 was not inhibited significantly (Fig. A , compare lane 3 with lanes 2 and 4).

Expressing:

Article Title: Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation
Article Snippet: .. The efficient IFN antagonism induced by Nipah V protein expression in the absence of observed STAT1 or STAT2 degradation is consistent with an alternate mode of action for this V protein. .. Previous studies of IFN antagonism by SV5 and HPIV2 demonstrated that a multiprotein complex that includes V protein and both STAT1 and STAT2 proteins is essential for IFN antagonism ( ).

Binding Assay:

Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?
Article Snippet: .. The specific interaction between Stat1 and CaMKII also suggests the possibility that CaMKII may phosphorylate sites in chromatin to participate in chromatin remodeling near the Stat1 binding sites, which may occur in addition to the acetylation of histones by CBP/p300, also recruited by Stat1 ( , ). ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Upstate Biotechnology Inc stat1
    DN forms of CaMKII inhibit <t>Stat1</t> S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
    Stat1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/stat1/product/Upstate Biotechnology Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    stat1 - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    85
    Upstate Biotechnology Inc anti phosphotyrosine 701 stat1
    Normal activation of <t>Stat1</t> by IFNγ in IKKα/β-null cells. WT or IKKα/β-null cells were treated for 15 or 30 min with IFNγ or were untreated. Western analysis was performed with antibodies against phosphoserine-727 Stat1, <t>phosphotyrosine-701</t> Stat1, and total Stat1.
    Anti Phosphotyrosine 701 Stat1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phosphotyrosine 701 stat1/product/Upstate Biotechnology Inc
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti phosphotyrosine 701 stat1 - by Bioz Stars, 2020-11
    85/100 stars
      Buy from Supplier

    80
    Upstate Biotechnology Inc rabbit anti human stat1 antibody
    Tyrosine phosphorylation of <t>Stat1,</t> Jak1, and Jak2 during IFN-γ stimulation in THP-1 cells. THP-1 cells (5 × 10 6 ) were incubated with 50 nM PMA for 18 h, washed twice with prewarmed RPMI 1640 medium, and stimulated with or without IFN-γ (1,000 U/ml) for 10 min. Cells were washed twice with prewarmed PBS and lysed in ice-cold lysis buffer. Cell lysates were immunoprecipitated with antibodies specific for Stat1, Jak1, Jak2, or normal rabbit serum (NS) as described in Materials and Methods. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis by using antiphosphotyrosine antibody. Stat1 (91 kDa), Jak1 (130 kDa), and Jak2 (130 kDa) are indicated. Data shown are from one of three independent experiments with similar results.
    Rabbit Anti Human Stat1 Antibody, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti human stat1 antibody/product/Upstate Biotechnology Inc
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti human stat1 antibody - by Bioz Stars, 2020-11
    80/100 stars
      Buy from Supplier

    89
    Upstate Biotechnology Inc antibodies against stat1
    LPMV-V protein inhibits Type I IFN induced phosphorylation of <t>STAT1</t> and STAT2 and prevents STAT1 and STAT2 nuclear translocation. (A) 293T cells were transfected with empty vector, LPMV-V-HA, NipahV-V-HA or PIV5-V-HA expression plasmids. Cells were treated for 30 min l with 1,000 U/ml of IFN β prior to lysis. Lysates were separated by SDS-PAGE (12 % polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1 STAT2 phosphotyrosine 701-STAT1 phosphotyrosine 690-STAT2, anti-actin and anti-HA epitope tag as describe in Materials and Methods. (B) 293T cells were transfected with empty vector, LPMV-V-HA, or PIV5-V-HA expression plasmids. Cells were treated with 1,000 U/ml of IFN β per 30 min prior to lysis. Lysates were separated by SDS-PAGE (12 % polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1 STAT2 phosphotyrosine 701-STAT1, phosphotyrosine 690-STAT2, anti-tubulin and anti-HA epitope tag as describe in Materials and Methods. (C) HeLa cells were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids. Before fixation, cells were treated for 30 minutes with 1000 U/ml of IFN β. Cells were fixed, permeabilized, and stained with antibodies against STAT1 and HA as described in Materials and Methods. (D) HeLa cells were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids. Before fixation, cells were treated for 30 minutes with 1000 U/ml of IFN β. Cells were fixed, permeabilized, and stained with antibodies to STAT2 and HA, as described in Materials and Methods. Nuclear DNA was stained with DAPI. Images were obtained using confocal microscopy.
    Antibodies Against Stat1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against stat1/product/Upstate Biotechnology Inc
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against stat1 - by Bioz Stars, 2020-11
    89/100 stars
      Buy from Supplier

    Image Search Results


    DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

    doi: 10.1073/pnas.052159099

    Figure Lengend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    Article Snippet: Altogether, these results demonstrate that there is a functional enzyme–substrate interaction between Stat1 and CaMKII.

    Techniques: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

    Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

    doi: 10.1073/pnas.052159099

    Figure Lengend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

    Article Snippet: Altogether, these results demonstrate that there is a functional enzyme–substrate interaction between Stat1 and CaMKII.

    Techniques: Activation Assay, Western Blot, Northern Blot

    CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

    doi: 10.1073/pnas.052159099

    Figure Lengend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

    Article Snippet: Altogether, these results demonstrate that there is a functional enzyme–substrate interaction between Stat1 and CaMKII.

    Techniques: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

    Normal activation of Stat1 by IFNγ in IKKα/β-null cells. WT or IKKα/β-null cells were treated for 15 or 30 min with IFNγ or were untreated. Western analysis was performed with antibodies against phosphoserine-727 Stat1, phosphotyrosine-701 Stat1, and total Stat1.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Inhibitor of ?B kinase is required to activate a subset of interferon ?-stimulated genes

    doi: 10.1073/pnas.0401593101

    Figure Lengend Snippet: Normal activation of Stat1 by IFNγ in IKKα/β-null cells. WT or IKKα/β-null cells were treated for 15 or 30 min with IFNγ or were untreated. Western analysis was performed with antibodies against phosphoserine-727 Stat1, phosphotyrosine-701 Stat1, and total Stat1.

    Article Snippet: Polyclonal anti-phosphoserine-727 Stat1, anti-phosphotyrosine-701 Stat1, and anti-Stat1 antibodies were from Upstate Biotechnology (Lake Placid, NY).

    Techniques: Activation Assay, Western Blot

    Tyrosine phosphorylation of Stat1, Jak1, and Jak2 during IFN-γ stimulation in THP-1 cells. THP-1 cells (5 × 10 6 ) were incubated with 50 nM PMA for 18 h, washed twice with prewarmed RPMI 1640 medium, and stimulated with or without IFN-γ (1,000 U/ml) for 10 min. Cells were washed twice with prewarmed PBS and lysed in ice-cold lysis buffer. Cell lysates were immunoprecipitated with antibodies specific for Stat1, Jak1, Jak2, or normal rabbit serum (NS) as described in Materials and Methods. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis by using antiphosphotyrosine antibody. Stat1 (91 kDa), Jak1 (130 kDa), and Jak2 (130 kDa) are indicated. Data shown are from one of three independent experiments with similar results.

    Journal: Infection and Immunity

    Article Title: Protein Kinase A-Mediated Inhibition of Gamma Interferon-Induced Tyrosine Phosphorylation of Janus Kinases and Latent Cytoplasmic Transcription Factors in Human Monocytes by Ehrlichia chaffeensis

    doi:

    Figure Lengend Snippet: Tyrosine phosphorylation of Stat1, Jak1, and Jak2 during IFN-γ stimulation in THP-1 cells. THP-1 cells (5 × 10 6 ) were incubated with 50 nM PMA for 18 h, washed twice with prewarmed RPMI 1640 medium, and stimulated with or without IFN-γ (1,000 U/ml) for 10 min. Cells were washed twice with prewarmed PBS and lysed in ice-cold lysis buffer. Cell lysates were immunoprecipitated with antibodies specific for Stat1, Jak1, Jak2, or normal rabbit serum (NS) as described in Materials and Methods. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis by using antiphosphotyrosine antibody. Stat1 (91 kDa), Jak1 (130 kDa), and Jak2 (130 kDa) are indicated. Data shown are from one of three independent experiments with similar results.

    Article Snippet: The supernatants containing 500 μg of protein in 500 μl were incubated for 16 to 18 h at 4°C with 4 μl of rabbit anti-human Stat1 antibody, rabbit anti-human Jak1 antiserum, rabbit anti-human Jak2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y.), or normal rabbit serum.

    Techniques: Incubation, Lysis, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot

    Time course of the effect of E. chaffeensis infection on tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-γ. THP-1 cells preincubated with PMA for 18 h were treated with host cell-free E. chaffeensis organisms for the indicated periods of time and stimulated with IFN-γ for 10 min. The cell lysates were immunoprecipitated with anti-Stat1, anti-Jak1, or anti-Jak2 antibody, and Western blot analysis was performed with antiphosphotyrosine antibody. Similar results were obtained in two experiments.

    Journal: Infection and Immunity

    Article Title: Protein Kinase A-Mediated Inhibition of Gamma Interferon-Induced Tyrosine Phosphorylation of Janus Kinases and Latent Cytoplasmic Transcription Factors in Human Monocytes by Ehrlichia chaffeensis

    doi:

    Figure Lengend Snippet: Time course of the effect of E. chaffeensis infection on tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-γ. THP-1 cells preincubated with PMA for 18 h were treated with host cell-free E. chaffeensis organisms for the indicated periods of time and stimulated with IFN-γ for 10 min. The cell lysates were immunoprecipitated with anti-Stat1, anti-Jak1, or anti-Jak2 antibody, and Western blot analysis was performed with antiphosphotyrosine antibody. Similar results were obtained in two experiments.

    Article Snippet: The supernatants containing 500 μg of protein in 500 μl were incubated for 16 to 18 h at 4°C with 4 μl of rabbit anti-human Stat1 antibody, rabbit anti-human Jak1 antiserum, rabbit anti-human Jak2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y.), or normal rabbit serum.

    Techniques: Infection, Immunoprecipitation, Western Blot

    Protein factors of E. chaffeensis are required for the inhibition of tyrosine phosphorylation of Stat1. THP-1 cells preincubated with PMA for 18 h were subjected to different treatment for 30 min followed by IFN-γ stimulation for 10 min. The cell lysates were immunoprecipitated with anti-Stat1 antibody and analyzed by immunoblotting. The upper panel represents the membrane probed with antiphosphotyrosine (α-PTyr), and the lower panel represents the membrane probed with anti-Stat1 (α-Stat1). Lanes: 1, untreated control cells without IFN-γ stimulation; 2 to 8, cells stimulated with IFN-γ; 2, untreated cells; 3, cells treated with viable E. chaffeensis ; 4, cells treated with heat-killed E. chaffeensis ; 5, cells treated with periodate-treated E. chaffeensis ; 6, cells treated with proteinase K-treated E. chaffeensis ; 7, cells treated with lysate of E. chaffeensis ; 8, cells treated with E. coli ). Two separate experiments yielded similar results.

    Journal: Infection and Immunity

    Article Title: Protein Kinase A-Mediated Inhibition of Gamma Interferon-Induced Tyrosine Phosphorylation of Janus Kinases and Latent Cytoplasmic Transcription Factors in Human Monocytes by Ehrlichia chaffeensis

    doi:

    Figure Lengend Snippet: Protein factors of E. chaffeensis are required for the inhibition of tyrosine phosphorylation of Stat1. THP-1 cells preincubated with PMA for 18 h were subjected to different treatment for 30 min followed by IFN-γ stimulation for 10 min. The cell lysates were immunoprecipitated with anti-Stat1 antibody and analyzed by immunoblotting. The upper panel represents the membrane probed with antiphosphotyrosine (α-PTyr), and the lower panel represents the membrane probed with anti-Stat1 (α-Stat1). Lanes: 1, untreated control cells without IFN-γ stimulation; 2 to 8, cells stimulated with IFN-γ; 2, untreated cells; 3, cells treated with viable E. chaffeensis ; 4, cells treated with heat-killed E. chaffeensis ; 5, cells treated with periodate-treated E. chaffeensis ; 6, cells treated with proteinase K-treated E. chaffeensis ; 7, cells treated with lysate of E. chaffeensis ; 8, cells treated with E. coli ). Two separate experiments yielded similar results.

    Article Snippet: The supernatants containing 500 μg of protein in 500 μl were incubated for 16 to 18 h at 4°C with 4 μl of rabbit anti-human Stat1 antibody, rabbit anti-human Jak1 antiserum, rabbit anti-human Jak2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y.), or normal rabbit serum.

    Techniques: Inhibition, Immunoprecipitation

    Effect of E. chaffeensis infection on tyrosine phosphorylation of Stat1, Jak1, and Jak2 in human peripheral blood monocytes. Human peripheral blood monocytes were treated with E. chaffeensis for 30 min followed by IFN-γ stimulation for 10 min. Cell lysates were immunoprecipitated with antibodies for Stat1, Jak1, or Jak2 and subjected to Western blot analysis. Lanes: 1, 4, and 7, untreated control cells without IFN-γ stimulation; 2, 5, and 8, untreated cells stimulated with IFN-γ; 3, 6, and 9, cells treated with E. chaffeensis and stimulated with IFN-γ. The data presented are from one of two independent experiments that gave similar results.

    Journal: Infection and Immunity

    Article Title: Protein Kinase A-Mediated Inhibition of Gamma Interferon-Induced Tyrosine Phosphorylation of Janus Kinases and Latent Cytoplasmic Transcription Factors in Human Monocytes by Ehrlichia chaffeensis

    doi:

    Figure Lengend Snippet: Effect of E. chaffeensis infection on tyrosine phosphorylation of Stat1, Jak1, and Jak2 in human peripheral blood monocytes. Human peripheral blood monocytes were treated with E. chaffeensis for 30 min followed by IFN-γ stimulation for 10 min. Cell lysates were immunoprecipitated with antibodies for Stat1, Jak1, or Jak2 and subjected to Western blot analysis. Lanes: 1, 4, and 7, untreated control cells without IFN-γ stimulation; 2, 5, and 8, untreated cells stimulated with IFN-γ; 3, 6, and 9, cells treated with E. chaffeensis and stimulated with IFN-γ. The data presented are from one of two independent experiments that gave similar results.

    Article Snippet: The supernatants containing 500 μg of protein in 500 μl were incubated for 16 to 18 h at 4°C with 4 μl of rabbit anti-human Stat1 antibody, rabbit anti-human Jak1 antiserum, rabbit anti-human Jak2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y.), or normal rabbit serum.

    Techniques: Infection, Immunoprecipitation, Western Blot

    LPMV-V protein inhibits Type I IFN induced phosphorylation of STAT1 and STAT2 and prevents STAT1 and STAT2 nuclear translocation. (A) 293T cells were transfected with empty vector, LPMV-V-HA, NipahV-V-HA or PIV5-V-HA expression plasmids. Cells were treated for 30 min l with 1,000 U/ml of IFN β prior to lysis. Lysates were separated by SDS-PAGE (12 % polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1 STAT2 phosphotyrosine 701-STAT1 phosphotyrosine 690-STAT2, anti-actin and anti-HA epitope tag as describe in Materials and Methods. (B) 293T cells were transfected with empty vector, LPMV-V-HA, or PIV5-V-HA expression plasmids. Cells were treated with 1,000 U/ml of IFN β per 30 min prior to lysis. Lysates were separated by SDS-PAGE (12 % polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1 STAT2 phosphotyrosine 701-STAT1, phosphotyrosine 690-STAT2, anti-tubulin and anti-HA epitope tag as describe in Materials and Methods. (C) HeLa cells were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids. Before fixation, cells were treated for 30 minutes with 1000 U/ml of IFN β. Cells were fixed, permeabilized, and stained with antibodies against STAT1 and HA as described in Materials and Methods. (D) HeLa cells were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids. Before fixation, cells were treated for 30 minutes with 1000 U/ml of IFN β. Cells were fixed, permeabilized, and stained with antibodies to STAT2 and HA, as described in Materials and Methods. Nuclear DNA was stained with DAPI. Images were obtained using confocal microscopy.

    Journal: Virus research

    Article Title: La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation

    doi: 10.1016/j.virusres.2015.10.027

    Figure Lengend Snippet: LPMV-V protein inhibits Type I IFN induced phosphorylation of STAT1 and STAT2 and prevents STAT1 and STAT2 nuclear translocation. (A) 293T cells were transfected with empty vector, LPMV-V-HA, NipahV-V-HA or PIV5-V-HA expression plasmids. Cells were treated for 30 min l with 1,000 U/ml of IFN β prior to lysis. Lysates were separated by SDS-PAGE (12 % polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1 STAT2 phosphotyrosine 701-STAT1 phosphotyrosine 690-STAT2, anti-actin and anti-HA epitope tag as describe in Materials and Methods. (B) 293T cells were transfected with empty vector, LPMV-V-HA, or PIV5-V-HA expression plasmids. Cells were treated with 1,000 U/ml of IFN β per 30 min prior to lysis. Lysates were separated by SDS-PAGE (12 % polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1 STAT2 phosphotyrosine 701-STAT1, phosphotyrosine 690-STAT2, anti-tubulin and anti-HA epitope tag as describe in Materials and Methods. (C) HeLa cells were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids. Before fixation, cells were treated for 30 minutes with 1000 U/ml of IFN β. Cells were fixed, permeabilized, and stained with antibodies against STAT1 and HA as described in Materials and Methods. (D) HeLa cells were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids. Before fixation, cells were treated for 30 minutes with 1000 U/ml of IFN β. Cells were fixed, permeabilized, and stained with antibodies to STAT2 and HA, as described in Materials and Methods. Nuclear DNA was stained with DAPI. Images were obtained using confocal microscopy.

    Article Snippet: Proteins were then transferred to nitrocellulose membranes and were probed with antibodies against STAT1 (Upstate Biotechnology, no. 06-501), STAT2 (Santa Cruz, no. sc-476), phosphotyrosine 701-STAT1 (Cell Signaling Technologies, no. 7649), Anti-phospho-STAT2 (Tyr689) (Millipore no: 07-224), anti-actin (Sigma–Aldrich # A 5060) anti β tubulin (Cell Signaling #2146) and anti-HA epitope tag (Sigma-Aldrich # H3663), and visualized by chemiluminescence (NEN Life Sciences).

    Techniques: Translocation Assay, Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Staining, Confocal Microscopy

    LPMV-V protein interacts with human and porcine STAT2. (A and B) LPMV-V-HA, NipahV-V-Ha Ha, PIV5-V-Ha plasmids and empty vector were transfected in 293T cells. At 28 hours postransfection, the cells were treated (or left untreated) with 1000 U/ml IFN-β for 24 hours at 37°C. 48 hours after transfection cells were lysate. Aliquots for total protein analyses were separated, and the remaining supernatants were incubated for 3 hours with the Red Anti-HA affinity gel antibody or without antibody. The pellets were washed six times with whole-cell extract buffer and finally dissolved directly in Laemmli sample buffer. The samples were analyzed on sodium dodecyl sulfate (SDS) 4–15% polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes and were probed with antibodies against STAT1, STAT2, HA and Actin. (C) Reciprocal co-immunoprecipitation in 293t cells using an anti-STAT2 antibody was performed. (D) Co-immunoprecipitation assay was performed in porcine cells PK-15.

    Journal: Virus research

    Article Title: La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation

    doi: 10.1016/j.virusres.2015.10.027

    Figure Lengend Snippet: LPMV-V protein interacts with human and porcine STAT2. (A and B) LPMV-V-HA, NipahV-V-Ha Ha, PIV5-V-Ha plasmids and empty vector were transfected in 293T cells. At 28 hours postransfection, the cells were treated (or left untreated) with 1000 U/ml IFN-β for 24 hours at 37°C. 48 hours after transfection cells were lysate. Aliquots for total protein analyses were separated, and the remaining supernatants were incubated for 3 hours with the Red Anti-HA affinity gel antibody or without antibody. The pellets were washed six times with whole-cell extract buffer and finally dissolved directly in Laemmli sample buffer. The samples were analyzed on sodium dodecyl sulfate (SDS) 4–15% polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes and were probed with antibodies against STAT1, STAT2, HA and Actin. (C) Reciprocal co-immunoprecipitation in 293t cells using an anti-STAT2 antibody was performed. (D) Co-immunoprecipitation assay was performed in porcine cells PK-15.

    Article Snippet: Proteins were then transferred to nitrocellulose membranes and were probed with antibodies against STAT1 (Upstate Biotechnology, no. 06-501), STAT2 (Santa Cruz, no. sc-476), phosphotyrosine 701-STAT1 (Cell Signaling Technologies, no. 7649), Anti-phospho-STAT2 (Tyr689) (Millipore no: 07-224), anti-actin (Sigma–Aldrich # A 5060) anti β tubulin (Cell Signaling #2146) and anti-HA epitope tag (Sigma-Aldrich # H3663), and visualized by chemiluminescence (NEN Life Sciences).

    Techniques: Plasmid Preparation, Transfection, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay