samhd1 (OriGene)

OriGene samhd1

Figure 1. Knockout of <t>SAMHD1</t> activates an immune response in human monocytic cells. (A,B) Relative mRNA levels for the indicated genes in SAMHD1-deficient cells, as assessed by qRT-PCR and normalized to β-actin expression. (C) ELISA of IFN-α production in cell extracts. ELISA of IFN-β production in supernatants. Conditioned media were concentrated using Amicon Ultra-15 before analysis. (D) qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and MxA in PMA-differentiated wild-type and SAMHD1-deficient THP-1 cells. (E) Wild-type and SAMHD1-deficient THP-1 cells were incubated in basal media containing 10% FBS or reduced-serum media containing 4% or 2% FBS for 48 h, followed by qRT-PCR analysis of IFITM1 and MxA mRNA levels. Data were standardized to β-actin. In (A–E), data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Samhd1, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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samhd1 - by Bioz Stars, 2026-09

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rabbit anti- samhd1 polyclonal antibody (Cusabio)

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Rabbit anti-Homo sapiens (Human) SAMHD1 Polyclonal Antibody

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rabbit anti-mouse samhd1 polyclonal antibody (Cusabio)

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Rabbit anti-Mouse Samhd1 Polyclonal Antibody

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samhd1 rabbit polyclonal antibody (OriGene)

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Rabbit polyclonal antibody to SAMHD1 SAM domain and HD domain 1

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Figure 1. Knockout of SAMHD1 activates an immune response in human monocytic cells. (A,B) Relative mRNA levels for the indicated genes in SAMHD1-deficient cells, as assessed by qRT-PCR and normalized to β-actin expression. (C) ELISA of IFN-α production in cell extracts. ELISA of IFN-β production in supernatants. Conditioned media were concentrated using Amicon Ultra-15 before analysis. (D) qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and MxA in PMA-differentiated wild-type and SAMHD1-deficient THP-1 cells. (E) Wild-type and SAMHD1-deficient THP-1 cells were incubated in basal media containing 10% FBS or reduced-serum media containing 4% or 2% FBS for 48 h, followed by qRT-PCR analysis of IFITM1 and MxA mRNA levels. Data were standardized to β-actin. In (A–E), data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Journal: Scientific reports

Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature.

doi: 10.1038/s41598-017-18308-8

Figure Lengend Snippet: Figure 1. Knockout of SAMHD1 activates an immune response in human monocytic cells. (A,B) Relative mRNA levels for the indicated genes in SAMHD1-deficient cells, as assessed by qRT-PCR and normalized to β-actin expression. (C) ELISA of IFN-α production in cell extracts. ELISA of IFN-β production in supernatants. Conditioned media were concentrated using Amicon Ultra-15 before analysis. (D) qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and MxA in PMA-differentiated wild-type and SAMHD1-deficient THP-1 cells. (E) Wild-type and SAMHD1-deficient THP-1 cells were incubated in basal media containing 10% FBS or reduced-serum media containing 4% or 2% FBS for 48 h, followed by qRT-PCR analysis of IFITM1 and MxA mRNA levels. Data were standardized to β-actin. In (A–E), data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Article Snippet: The chemical reagents and antibodies used in this study were purchased from the following manufacturers: poly (I:C) and poly (dA:dT), Sigma; T4 Polynucleotide Kinase and T4 RNA ligase, Takara; T4 RNA Ligase 2 (truncated K227Q) and Antarctic Phosphatase, New England Biolabs (NEB); wortmannin and rapamycin, Sigma; MK-2206, A-674563, and Lapatinib, Selleckchem; Ruxolitinib, Invivogen; mouse monoclonal antibodies to IFNAR1 (Millipore) and SAMHD1 (OriGene); rabbit monoclonal antibodies to IRF3 pho-S386 (Abcam), RIG-I, STING, MyD88, TBK1, TBK1 pho-S172, Stat1 pho-Y701, and GSK-3β (all from Cell Signaling Technology); rabbit polyclonal antibodies to IRF3 (Santa Cruz), SAMHD1 (for immunoprecipitation, Bethyl Lab), GAPDH (Ab Frontier), MAVS, TRIF, IRF7, STAT1, Akt, Akt pho-S473, and GSK-3β pho-S9 (all from Cell Signaling Technology); goat polyclonal antibody to hIL-10Rb (R&D systems).

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Two Tailed Test

Figure 2. Identification and classification of DEGs in SAMHD1-deficient THP-1 cells. (A) RNA-seq MAplot of wild-type versus SAMHD1 knockout cells as indicated. Three biological replicates were analyzed for both data sets. Average gene expression is plotted on the x-axis and log2 fold-change is plotted on the y-axis; red dots: upregulated genes (log2 FC ≥ 1 and adjusted p-values < 0.01), green dots: downregulated genes (log2 FC ≤ −1 and adjusted p-values < 0.01), blue dots: ISGs. (B) Statistically significant signaling pathways for genes upregulated by over 2-fold in SAMHD1 knockout samples were obtained by Ingenuity Pathway Analysis (IPA). Blue bars indicate the ratio of the total number of genes involved in the specific pathway versus input list genes, while the orange squares show −log (p-value). (C) Heatmap of ISGs expressed in the indicated cells with the RNA-seq data. Gene expression levels (averaged reads per kilobase per million mapped reads (RPKM) values over 3 replicates) was standardized and clustered based on the dissimilarity values (1-Pearson correlation) between genes using the average linkage method as shown in the dendrogram. (D) The mRNA levels of ISGs in wild-type and SAMHD1-deficient THP-1 cells were determined by qRT-PCR. Data were normalized to the GAPDH level. Data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, two-tailed Student’s t-test).

Journal: Scientific reports

Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature.

doi: 10.1038/s41598-017-18308-8

Figure Lengend Snippet: Figure 2. Identification and classification of DEGs in SAMHD1-deficient THP-1 cells. (A) RNA-seq MAplot of wild-type versus SAMHD1 knockout cells as indicated. Three biological replicates were analyzed for both data sets. Average gene expression is plotted on the x-axis and log2 fold-change is plotted on the y-axis; red dots: upregulated genes (log2 FC ≥ 1 and adjusted p-values < 0.01), green dots: downregulated genes (log2 FC ≤ −1 and adjusted p-values < 0.01), blue dots: ISGs. (B) Statistically significant signaling pathways for genes upregulated by over 2-fold in SAMHD1 knockout samples were obtained by Ingenuity Pathway Analysis (IPA). Blue bars indicate the ratio of the total number of genes involved in the specific pathway versus input list genes, while the orange squares show −log (p-value). (C) Heatmap of ISGs expressed in the indicated cells with the RNA-seq data. Gene expression levels (averaged reads per kilobase per million mapped reads (RPKM) values over 3 replicates) was standardized and clustered based on the dissimilarity values (1-Pearson correlation) between genes using the average linkage method as shown in the dendrogram. (D) The mRNA levels of ISGs in wild-type and SAMHD1-deficient THP-1 cells were determined by qRT-PCR. Data were normalized to the GAPDH level. Data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, two-tailed Student’s t-test).

Techniques: RNA Sequencing, Knock-Out, Gene Expression, Protein-Protein interactions, Quantitative RT-PCR, Two Tailed Test

$Figure 3. Accumulated RNAs in SAMHD1-deficient cells function as immune stimuli. (A,B) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1-deficient cells, or left unstimulated (A). Total RNA isolated from wild-type and SAMHD1-deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells (B), followed by qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and IL6 mRNA levels. (C,D) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs (D). (E) qRT-PCR analysis of IFN-α in wild-type and SAMHD1- deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. (F) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. (G) Pie chart showing the distribution of statistically significant peaks (q < 0.001) among the indicated RNA classes. Data were normalized to β-actin expression. In (A), (B) and (E), these data represent the mean ± SEM of triplicate independent experiments (**p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).$

Journal: Scientific reports

Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature.

doi: 10.1038/s41598-017-18308-8

Figure Lengend Snippet: Figure 3. Accumulated RNAs in SAMHD1-deficient cells function as immune stimuli. (A,B) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1-deficient cells, or left unstimulated (A). Total RNA isolated from wild-type and SAMHD1-deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells (B), followed by qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and IL6 mRNA levels. (C,D) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs (D). (E) qRT-PCR analysis of IFN-α in wild-type and SAMHD1- deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. (F) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. (G) Pie chart showing the distribution of statistically significant peaks (q < 0.001) among the indicated RNA classes. Data were normalized to β-actin expression. In (A), (B) and (E), these data represent the mean ± SEM of triplicate independent experiments (**p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Techniques: Isolation, Quantitative RT-PCR, In Vitro, Activity Assay, Control, Immu-Puri, Infection, Serial Dilution, Mutagenesis, Construct, Autoradiography, Western Blot, Immunoprecipitation, Expressing, Two Tailed Test

Figure 4. ISGs induction in SAMHD1-deficiency occurs through IRF3 and type I IFN receptor signaling pathway. (A,B) Wild-type and SAMHD1-deficient THP-1 cells were transfected with control siRNA or specific siRNA for the indicated genes for 72 h. Cell lysates were subjected to western blotting to analyze the protein levels. GAPDH was loaded as a control (A). The cellular mRNA was analyzed by qRT-PCR to determine the expression of IFITM1 and MxA relative to GAPDH (B). (C) qRT-PCR analysis of IFN-α and MxA in THP-1 cells incubated with conditioned media (Cond. Media.) from wild-type or SAMHD1-deficient cells for the indicated days. (D) qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and MxA in wild-type and SAMHD1-deficient THP-1 cells treated with control IgG or neutralizing antibodies against type I and III IFN receptor subunits as indicated. Results were standardized to β-actin levels. (E) The measurement of IFITM1 and MxA mRNA expressions in SAMHD1-deficient cells by qRT-PCR after treating SAMHD1-deficient cells with control dimethylsulfoxide (DMSO) or 2 µM Ruxolitinib for 24 h. Data were standardized to GAPDH. In (B–E), these data represent the mean ± SEM of triplicate independent experiments. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Journal: Scientific reports

Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature.

doi: 10.1038/s41598-017-18308-8

Figure Lengend Snippet: Figure 4. ISGs induction in SAMHD1-deficiency occurs through IRF3 and type I IFN receptor signaling pathway. (A,B) Wild-type and SAMHD1-deficient THP-1 cells were transfected with control siRNA or specific siRNA for the indicated genes for 72 h. Cell lysates were subjected to western blotting to analyze the protein levels. GAPDH was loaded as a control (A). The cellular mRNA was analyzed by qRT-PCR to determine the expression of IFITM1 and MxA relative to GAPDH (B). (C) qRT-PCR analysis of IFN-α and MxA in THP-1 cells incubated with conditioned media (Cond. Media.) from wild-type or SAMHD1-deficient cells for the indicated days. (D) qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and MxA in wild-type and SAMHD1-deficient THP-1 cells treated with control IgG or neutralizing antibodies against type I and III IFN receptor subunits as indicated. Results were standardized to β-actin levels. (E) The measurement of IFITM1 and MxA mRNA expressions in SAMHD1-deficient cells by qRT-PCR after treating SAMHD1-deficient cells with control dimethylsulfoxide (DMSO) or 2 µM Ruxolitinib for 24 h. Data were standardized to GAPDH. In (B–E), these data represent the mean ± SEM of triplicate independent experiments. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Expressing, Incubation, Two Tailed Test

Figure 5. The PI3K/AKT pathway is responsible for linking SAMHD1-deficiency to the IFN response. (A) Western blotting analysis of cell extracts from wild-type and two independent SAMHD1-deficient cell lines using the indicated antibodies. (B) qRT-PCR analysis of IFN-α, IFN-β and MxA levels in wild-type and SAMHD1-deficient THP-1 cells treated with control DMSO or 1 µM Wortmannin for 24 h. Data were normalized to GAPDH levels. (C,D) Wild-type and SAMHD1-deficient THP-1 cells were treated with control DMSO, 1 µM MK2206, 1 µM A674563, 0.1 µM Lapatinib or 50 nM Rapamycin for 24 h. Cells were analyzed for the mRNA levels of IFN-α, IFN-β and MxA relative to GAPDH by qRT-PCR (C) and for the western blotting analysis to determine the phosphorylation status of STAT1, AKT and IRF3 (D). GAPDH served as a loading control. In (B) and (C), these data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Journal: Scientific reports

Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature.

doi: 10.1038/s41598-017-18308-8

Figure Lengend Snippet: Figure 5. The PI3K/AKT pathway is responsible for linking SAMHD1-deficiency to the IFN response. (A) Western blotting analysis of cell extracts from wild-type and two independent SAMHD1-deficient cell lines using the indicated antibodies. (B) qRT-PCR analysis of IFN-α, IFN-β and MxA levels in wild-type and SAMHD1-deficient THP-1 cells treated with control DMSO or 1 µM Wortmannin for 24 h. Data were normalized to GAPDH levels. (C,D) Wild-type and SAMHD1-deficient THP-1 cells were treated with control DMSO, 1 µM MK2206, 1 µM A674563, 0.1 µM Lapatinib or 50 nM Rapamycin for 24 h. Cells were analyzed for the mRNA levels of IFN-α, IFN-β and MxA relative to GAPDH by qRT-PCR (C) and for the western blotting analysis to determine the phosphorylation status of STAT1, AKT and IRF3 (D). GAPDH served as a loading control. In (B) and (C), these data represent the mean ± SEM of triplicate independent experiments (*p ≤ 0.05, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).

Techniques: Western Blot, Quantitative RT-PCR, Control, Phospho-proteomics, Two Tailed Test

Figure 6. The PI3K/AKT functions upstream of IRF3 to activate type I IFN response. (A) The phosphorylation status of STAT1 and IRF3 in wild-type, SAMHD1 knockout and SAMHD1/AKT double-knockout THP-1 cells was determined by western blotting analysis. (B,C) Western blotting analysis of cell extracts from wild- type and SAMHD1-deficient cells treated with control IgG or a neutralizing antibody against a type I IFN receptor subunit for 48 h (B) or transfected with control non-specific siRNA or a specific siRNA for IRF3 (C). (D) Determination of the phosphorylation status of AKT after reconstitution of SAMHD1-deficient cells with wild-type and SAMHD1 mutants by western blotting analysis. Graph shown below indicates the ratio of phosphoAKT to wild-type control. (E) Isolated PBMCs from four donors were transfected with SAMHD1- specific siRNA or control non-specific siRNA for two cycles to enhance the knockdown efficiency. After 48 h of incubation, PBMCs were analyzed by western blotting to monitor the activation of STAT1 and AKT. Data are representative of four independent experiments with similar results. (F,G) PBMCs were infected with Vpx-loaded or control SIV VLPs. After 96 h of incubation, cells were analyzed for the western blotting analysis to determine the phosphorylation status of STAT1 and AKT (F) and for the mRNA levels of IFITM1, MxA and IFI44L relative to GAPDH by qRT-PCR (G). In (A–F), GAPDH served as a loading control. In (G), data represent the mean ± SEM of triplicate independent experiments (***p ≤ 0.001, two-tailed Student’s t-test).

Journal: Scientific reports

Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature.

doi: 10.1038/s41598-017-18308-8

Figure Lengend Snippet: Figure 6. The PI3K/AKT functions upstream of IRF3 to activate type I IFN response. (A) The phosphorylation status of STAT1 and IRF3 in wild-type, SAMHD1 knockout and SAMHD1/AKT double-knockout THP-1 cells was determined by western blotting analysis. (B,C) Western blotting analysis of cell extracts from wild- type and SAMHD1-deficient cells treated with control IgG or a neutralizing antibody against a type I IFN receptor subunit for 48 h (B) or transfected with control non-specific siRNA or a specific siRNA for IRF3 (C). (D) Determination of the phosphorylation status of AKT after reconstitution of SAMHD1-deficient cells with wild-type and SAMHD1 mutants by western blotting analysis. Graph shown below indicates the ratio of phosphoAKT to wild-type control. (E) Isolated PBMCs from four donors were transfected with SAMHD1- specific siRNA or control non-specific siRNA for two cycles to enhance the knockdown efficiency. After 48 h of incubation, PBMCs were analyzed by western blotting to monitor the activation of STAT1 and AKT. Data are representative of four independent experiments with similar results. (F,G) PBMCs were infected with Vpx-loaded or control SIV VLPs. After 96 h of incubation, cells were analyzed for the western blotting analysis to determine the phosphorylation status of STAT1 and AKT (F) and for the mRNA levels of IFITM1, MxA and IFI44L relative to GAPDH by qRT-PCR (G). In (A–F), GAPDH served as a loading control. In (G), data represent the mean ± SEM of triplicate independent experiments (***p ≤ 0.001, two-tailed Student’s t-test).

Techniques: Phospho-proteomics, Knock-Out, Double Knockout, Western Blot, Control, Transfection, Isolation, Knockdown, Incubation, Activation Assay, Infection, Quantitative RT-PCR, Two Tailed Test

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