rabbit anti sp110  (Proteintech)

Journal: eLife

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

doi: 10.7554/eLife.67290

Figure Lengend Snippet: ( A ) BMMs were treated with 10 U/ml of IFNγ for 24 hr and cells were lysed with RIPA buffer. Five micrograms of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp110 –/– mice were analyzed (denoted lines 61, 65, and 71). ( B–D ) Lungs of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H&E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ), or monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 –/– lines are shown. Representative of two experiments ( B, D ); combined results of three infections ( C ). *p≤0.05; **p≤0.01; ***p≤0.005. BMM, bone marrow-derived macrophage; CFU, colony-forming unit; RIPA, radioimmunoprecipitation assay.

Article Snippet: Antibody , Rabbit polyclonal anti-mouse SP110 (serum) , Covance, this paper , , WB (1:1000).

Techniques: Western Blot, Molecular Weight, Infection, Staining, MANN-WHITNEY, Derivative Assay, Radio Immunoprecipitation

Journal: eLife

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

doi: 10.7554/eLife.67290

Figure Lengend Snippet: ( A ) Mouse Sp110 gene. Guide RNA sequence for CRISPR/Cas9 targeting and protospacer-adjacent motif (PAM) are indicated. ( B–D ) Sp110 locus in wild-type (WT) and three independent lines. Homozygotes of two lines identified by sequencing ( B, C ), and heterozygote of the third line by PCR products separated on an agarose gel ( D ). Arrow indicates the mutant band. PCR, polymerase chain reaction.

Article Snippet: Antibody , Rabbit polyclonal anti-mouse SP110 (serum) , Covance, this paper , , WB (1:1000).

Techniques: Sequencing, CRISPR, Agarose Gel Electrophoresis, Mutagenesis, Polymerase Chain Reaction

Journal: eLife

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

doi: 10.7554/eLife.67290

Figure Lengend Snippet: ( A ) RT-PCR of cDNA from BMMs of the indicated genotypes. Red arrow indicates band corresponding to a portion of Sp140 , verified by sequencing. ( B ) Immunoblot of lysates from Sp140 –/– and B6 BMMs treated with 10 U/ml of recombinant mouse IFNγ for 24 hr. Equal amounts of protein were loaded for immunoblot with anti-SP140 antibody. ( C–F ) Mice were infected with Mycobacterium tuberculosis and measured for ( C ) lung CFU at 28 days post-infection, ( E ) body weight over time, and ( F ) survival. Statistics in ( E ) shows the comparison to B6 at day 28, and data are from 10 B6, 11 B6. Sst1 S , 11 Sp110 –/– , 14 Sp140 –/– , and 6 Sp140 +/– mice. ( D ) H&E staining of lungs at 25 days post-infection with M. tuberculosis . Full histology images are provided in . ( G ) Mice were infected with Legionella pneumophila and lung CFUs were determined at 96 hr post-infection. All mice were bred in-house, Sp140 –/– and Sp140 +/– were littermates ( C–F ). ( C ), ( E ), and ( G ) are combined results of two independent infections. ( A–D ) show representative analysis of one Sp140 –/– line (line 1), whereas ( F, G ) include a mixture of both lines 1 and 2. Results of infection of both lines with M. tuberculosis are shown in . ( C, E, F, G ) Mann-Whitney test. *p≤0.05; **p≤0.01; ***p≤0.005. BMM, bone marrow-derived macrophage; CFU, colony-forming unit; H&E, hematoxylin and eosin; RT-qPCR, real-time quantitative-polymerase chain reaction; WT, wild-type.

Article Snippet: Antibody , Rabbit polyclonal anti-mouse SP110 (serum) , Covance, this paper , , WB (1:1000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Recombinant, Infection, Comparison, Staining, MANN-WHITNEY, Derivative Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

doi: 10.7554/eLife.67290

Figure Lengend Snippet: ( A ) Mouse Sp140 gene. Guide RNA sequence for CRISPR/Cas9 targeting and protospacer-adjacent motif (PAM) are indicated. ( B, C ) Sp140 locus in wild-type (WT) and two independent founders of Sp140 –/– validated by sequencing. ( D ) Immunoblot for SP110 using BMMs from mice of the indicated genotypes. Intervening lanes have been removed for clarity (indicated by line in the image). ( E ) Mycobacterium tuberculosis -infected mice were harvested for CFU at 25 days post-infection. Empty and filled triangles indicate the two independent lines of Sp140 –/– used in this infection. All mice were bred in-house and Sp140 +/– ± littermates with Sp140 –/– line 2. Mann-Whitney test. *p≤0.05; **p≤0.01; ***p≤0.005. BMM, bone marrow-derived macrophage; CFU, colony-forming unit.

Article Snippet: Antibody , Rabbit polyclonal anti-mouse SP110 (serum) , Covance, this paper , , WB (1:1000).

Techniques: Sequencing, CRISPR, Western Blot, Infection, MANN-WHITNEY, Derivative Assay

Journal: eLife

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

doi: 10.7554/eLife.67290

Figure Lengend Snippet: Log 2 -fold change and adjusted p-value for SP family members ( Sp100, Sp110, and Sp140 ) from RNA-seq of M. tuberculosis -infected lungs from Sp110 –/– and Sp140 –/– mice, compared to B6 and B6. Sst1 S .

Article Snippet: Antibody , Rabbit polyclonal anti-mouse SP110 (serum) , Covance, this paper , , WB (1:1000).

Techniques: RNA Sequencing, Infection

Journal: eLife

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

doi: 10.7554/eLife.67290

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-mouse SP110 (serum) , Covance, this paper , , WB (1:1000).

Techniques: Cell Culture, Control, Injection, Recombinant, Plasmid Preparation, Derivative Assay, Sequencing

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: Upregulation of SP110 upon microbe infection or molecular treatment. (A) The expression of SP110 in an HBV-associated acute liver failure group was higher than in a normal-liver group. The values of HBV-associated acute liver failure groups were normalized to the mean of the normal-liver group (+SD). The GEO profile number is GDS4387 (GPL570 platform no. 209761_s_at). The data set title is “Hepatitis B virus (HBV)-associated acute liver failure (ALF) patients: liver explant” (58). **, P < 0.01. (B) Sendai virus infection effect on SP110 induction in a monocytic cell line. The values of Sendai virus-infected groups were normalized to the mean of the uninfected (UI) group (+SD). The GEO profile number is GDS6082 (GPL570 platform no. ILMN_2415144). The data set title is “Sendai virus infection effect on monocytic cell line: dose response” (59). (C) Pandemic and seasonal influenza virus H1N1 infection effect on SP110 induction in bronchial epithelial cells. The values of various H1N1 strain-infected groups were normalized to the mean of the UI group (+SD). The GEO profile number is GDS4855 (GPL570 platform no. 208012_x_at). The data set title is “Pandemic and seasonal H1N1 influenza virus infections of bronchial epithelial cells in vitro” (60). (D) Effect of H37Rv infection on SP110 induction in THP-1 cells. The values of the H37Rv-treated group were normalized to the mean of the UI group (+SD). The GEO profile number is GDS4781 (GPL570 platform no. 8059650). The data set headline is “Mycobacterium tuberculosis H37Rv-infected macrophage response to vitamin D” (61). (E) Effect of Mycobacterium bovis BCG infection on SP110 induction in THP-1 cells. The values of the BCG-treated group were normalized to the mean of the UI group (+SD). The GEO profile number is GDS2180 (GPL570 platform no. AGhsA070521). The data set title is “M-CSF and GM-CSF differentiated macrophage response to bacillus Calmette-Guerin (subset A)” (62). (F) The expression of SP110 in the etoposide-treated group was higher than that in the negative-control (NC) group. The values of the etoposide-treated group were normalized to the mean of the NC group (+SD). The GEO profile number is GDS5809 (GPL570 platform no. 8059650). The data set heading is “Anti-leukemia drug etoposide effect on ecotropic viral integration site 1-overexpressing myeloid cells” (63). (G) Cytarabine treatment leading to SP110 induction. The values of the DMSO-, doxorubicin-, puromycin-, and cytarabine-treated group were normalized to the mean of the DMSO (24-h) group (+SD). The GEO profile number is GDS2733 (GPL570 platform no. 208012_x_at). The data set headline is “Cytosine arabinoside effect on Ewing’s sarcoma cell line: time course and dose response” (64).

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Infection, Expressing, In Vitro, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: ISD induces Ipr1 expression through its promoter. (A) ISD activates Ipr1 expression in murine macrophages (RAW264.7 and J774A.1 cells), BMDMs, and fibroblasts (NIH 3T3 cells). The cells were transfected with ISD (2 μg/ml). After 36 h, the expression of Ipr1 was determined by qRT-PCR analysis. The values of ISD+ groups were normalized to the mean of ISD− groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (B) ISD induced Ipr1 expression in RAW264.7 cells and BMDMs by Western blotting in a time gradient. RAW264.7 cells and BMDMs were transfected with ISD for the indicated time intervals, and then protein levels were determined using anti-SP110 and anti-GAPDH by Western blotting. (C) ISD upregulates transcriptional activity of Ipr1 promoter reporter constructs in RAW264.7 and J774A.1 cells. Cells were transfected with promoter reporter constructs and the pGL4.73 vector for 24 h, and then ISD was transfected and the cells were incubated for 8 h. The transcriptional activity of reporter constructs was determined by luciferase assays. Values were normalized to the mean of the pGL4.10 (ISD−) group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (D) ISD activates luciferase activities of pathway reporter constructs ISRE-luc and NF-κB-luc. RAW264.7 cells were transfected with reporter plasmids pISRE-luc, pNF-κB-luc, pSRE-luc, pNFAT-luc, and pGRE-luc. After 24 h of transfection, the cells were transfected with 2 μg/ml ISD for 8 h, and then, the activity (+SD) of reporter constructs was determined by luciferase assays. The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (E) Homology analysis of the Ipr1 promoter region in different species. The different species’ Ipr1 promoter regions were downloaded from the UCSC database, and the sequence alignment was done with DNAMAN. (F) Classified analysis of transcription factors in different species’ Ipr1 promoters. The transcription factors of various species’ Ipr1 promoters were predicted using the JASPAR database with a relative profile score threshold of 80%. The clustering analysis used the online tool Venny 2.1.0 (bioinfogp.cnb.csic.es/tools/venny). (G) ISD activates SP110 expression in THP-1 cells. THP-1 cells were transfected with ISD for 24 h. The expression of Ipr1 was determined by qRT-PCR analysis (t test; **, P < 0.01).

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay, Construct, Plasmid Preparation, Incubation, Luciferase, Sequencing

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: Knockdown of Ipr1 decreases Irgm1 transcription. (A) Knockdown of Ipr1 downregulates Irgm1. siIpr1-1, siIpr1-2, and siNC were transfected into RAW264.7 cells with or without ISD treatment. The mRNAs were harvested after 36 h, and expression of Ipr1 and Irgm1 was examined by qRT-PCR. Values were normalized to the mean of the siNC ISD− group (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (B) Knockdown of Ipr1 downregulates Irgm1 at the protein level. siIpr1-1, siIpr1-2, and siNC were transfected into RAW264.7 cells with or without ISD, and the total protein was harvested after 48 h and detected with anti-SP110, anti-LRG47, and anti-GAPDH by Western blotting. (C) Knockdown of Ipr1 decreases transcriptional activity of the Irgm1 promoter. Irgm1 promoter reporter plasmids were cotransfected with siIpr1-1 or siIpr1-2 for 48 h, and the transcriptional activities were examined by luciferase assays. Values were normalized to the mean of the siNC-treated groups (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (D) Ipr1 enhances the transcriptional activity of the Irgm1 promoter. Irgm1 promoter reporter plasmids were cotransfected with pEGFP-C1 or pEGFP-Ipr1 into RAW264.7 and J774A.1 cells for 36 h, and then, the transcriptional activities were examined by luciferase assays. The Tnf-α promoter reporter plasmid was used as a positive control. The values of the pEGFP-Ipr1 groups were normalized to the mean of the pEGFP-C1 groups (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (E) Downregulation of Irgm1 and p21 after Ipr1 knockdown. RAW264.7 cells were transfected with siIpr1-1 and siNC for 36 h, and then the cells were treated with ISD for 8 h. The expression was determined by qRT-PCR. Values were normalized to the mean of those of the siNC (ISD−) groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (**, P < 0.01). (F) Proportion (percent) of G0/G1 cells (t test; **, P < 0.01).

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Plasmid Preparation, Positive Control

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: The p53 motif plays a critical role in Irgm1 transcriptional activation. (A) Mutation of the p53 motif impairs Irgm1 promoter transcription activity. RAW264.7 cells were transfected with the luciferase reporter plasmid Irgm1-PRO-900 and the mutated plasmid Irgm1-P900-p53mut. After 36 h of transfection, Act D and ISD were added to the culture medium for 8 h. Then, the transcriptional activity was determined by luciferase assay. Values were normalized to the mean of those in the Irgm1-PRO-900-DMSO group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (B) The p53 motif plays an important role in the Irgm1 promoter. The gene expression plasmid pCMV-p53 was cotransfected with the reporter plasmids Irgm1-PRO-900 and Irgm1-P900-p53mut for 36 h. Promoter transcriptional activity was determined by luciferase assay. Values were normalized to the mean of those in the Irgm1-PRO-900-pCMV-myc group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (C) pCMV-p53 or pCMV-myc was cotransfected with siNC or siIpr1-1 into RAW264.7 cells. After 48 h, cells were harvested and analyzed by Western blotting with anti-SP110 for IPR1 and anti-myc for myc-P53. Ipr1 siRNA, but not siNC, reduced the expression of Ipr1 successfully. (D) p53 rescued Irgm1 expression that was decreased by siIpr1-1. siNC, siIpr1-1, pCMV-myc, and pCMV-p53 were co-transfected into RAW264.7 cells for 36 h. The expression of Irgm1 was determined by qRT-PCR. Values were normalized to the mean of the siNC+ pCMV-myc group (+SD). The statistical significance of differences in relative mRNA expression was assessed by one-way ANOVA (**, P < 0.01).

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Activation Assay, Mutagenesis, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: IPR1 interacted with RPL11, MDM2, and NPM1. (A) Knockdown of p53 downregulates Irgm1 in protein. sip53-1, sip53-2, and siNC were transfected into RAW264.7 cells with or without ISD. Cells were harvested after 72 h and detected by anti-P53, anti-LRG47, and anti-GAPDH via Western blotting. (B) Knockdown of Ipr1 downregulates p53 in protein. siIpr1-1, siIpr1-2, and siNC were transfected into RAW264.7 cells with or without ISD. Cells were harvested after 72 h and detected by anti-P53, anti-SP110, and anti-GAPDH via Western blotting. (C) Protein-protein interaction network among IPR1, P53, and MDM2. P53 and MDM2 interactomes were obtained from BioGRID; the IPR1 interactome was determined in previous research. The interaction network was established with STRING. (D) RPL11 and NPM1 were common proteins in the P53 (Trp53), MDM2, and IPR1 interactome. The interactomes were clustered using Venny 2.1.0. (E) IPR1 interacted with RPL11, NPM1, and MDM2. EGFP-Ipr1 was coexpressed with Flag-Npm1, Flag-Rpl11, or HA-Mdm2 in HEK293T cells for 48 h. Anti-Flag or anti-HA was used for immunoprecipitation and anti-GFP for Western blotting (IB). (F) Live imaging of RAW264.7 cells expressing pEGFP-Ipr1 with mRuby, mRuby-H2b, mRuby-Mdm2, mRuby-Rpl11, or mRuby-Npm1. Scale bar, 10 μm.

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Transfection, Western Blot, Immunoprecipitation, Imaging, Expressing

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: IPR1 promotes P53 release by increasing interaction between RPL11 and MDM2, and the model of Ipr1 functions in signal transduction from ISD to Irgm1. (A) IPR1 enhanced interaction between RPL11 and MDM2. Flag-Rpl11 and HA-Mdm2 were transiently coexpressed in HEK293T cells with pEGFP-Ipr1 (lanes ii) or pEGFP-C1 (lanes i) for 48 h, and interactions were detected using anti-Flag for co-IP and anti-HA and anti-GFP for Western blotting. (B) IPR1 slightly decreased interaction between P53 and MDM2. Flag-p53 and HA-Mdm2 were transiently coexpressed in HEK293T cells with pEGFP-Ipr1 (lanes ii) or pEGFP-C1 (lanes i) for 48 h, and interactions were detected using anti-HA for co-IP and anti-Flag and anti-GFP for Western blotting. (C) IPR1 increased interaction between RPL11 and MDM2 and decreased interaction between P53 and MDM2 significantly. Flag-p53, Flag-Rpl11, and HA-Mdm2 were transiently coexpressed in HEK293T cells with pEGFP-Ipr1 (lanes ii) or pEGFP-C1 (lanes i) for 48 h, and interactions were detected using anti-HA for co-IP and anti-Flag and anti-GFP for Western blotting. (D) Knockdown of Rpl11 downregulates p53. siRpl11-1, siRpl11-2, and siNC were transfected into RAW264.7 cells with or without ISD treatment. Cells were harvested after 72 h and detected by anti-P53, anti-Rpl11, and anti-GAPDH by Western blotting. (E) ISD enhanced interaction between RPL11/IPR1 and MDM2. RAW264.7 cells were treated with ISD (lanes ii) or not (lanes i) for 60 h. Anti-MDM2 antibody was used for immunoprecipitation, and anti-Rpl11 and anti-SP110 were used for Western blotting. (F) Ubiquitination status of Flag-P53. Flag-p53, Flag-Rpl11, and HA-Mdm2 were cotransfected into HEK293T cells with pEGFP-Ipr1 or pEGFP-C1 for 60 h; 4 h prior to harvesting, cells were treated or not with MG132 (50 μM). Lysates were subjected to IP using anti-Flag. Bound proteins were analyzed by Western blotting using anti-Flag, anti-HA, anti-GFP, or anti-GAPDH. (G) Effects of ISD and Act D on THP-1 cells. THP-1 cells were treated with ISD or Act D for 16 h. Transcription of SP110, IRGM, and p21 was determined by qRT-PCR. The values were normalized to the mean of those in the ISD− or DMSO group (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (**, P < 0.01). (H) Effect of ISD on THP-1 cells. THP-1 cells were treated with ISD for 24 h. The proteins were determined by Western blotting using anti-SP110, anti-LRG47, and anti-GAPDH. (I) Model of Ipr1 functions in signal transduction from ISD to Irgm1. First, ISD is transfected into cells and arrested by cGAS. Then, the signals are transferred through cGAS-STING-TBK1 to IRF3, resulting in Ipr1 transcriptional activation. Afterward, IPR1 increases the release of P53 by promoting the interaction of RPL11 and MDM2. The released P53 regulates Irgm1 expression through the p53 motif in the Irgm1 promoter.

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Transduction, Co-Immunoprecipitation Assay, Western Blot, Transfection, Immunoprecipitation, Quantitative RT-PCR, Expressing, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53

doi: 10.1128/MCB.00471-19

Figure Lengend Snippet: qRT-PCR primers of human used in this study

Article Snippet: The membranes were incubated with one or more of the following primary antibodies: monoclonal rabbit anti-myc antibody (1:500; Beyotime; AM933), mouse monoclonal anti-Flag M2 (1:1,000; F1804; Sigma, USA), mouse monoclonal anti-LRG47 (1:100; sc-517338; Santa Cruz, USA), mouse monoclonal anti-green fluorescent protein (anti-GFP) (1:1,000; 66002-1-Ig; Proteintech, USA), rabbit polyclonal anti-Sp110 (M-190) (1:200; sc-98365; Santa Cruz, USA), and mouse polyclonal anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (1:1,000; A01622; TransGen, China).

Techniques: Sequencing