SAMHD1 (phospho Thr592) Antibody

Product Code: PSI-8005

Supplier: ProSci

Code	Size	Price

PSI-8005-0.02mg

0.02mg

£150.00

Quantity:

PSI-8005-0.1mg

0.1mg

£449.00

Quantity:

Prices exclude any Taxes / VAT

Overview

Host Type: Rabbit

Antibody Isotype: IgG

Antibody Clonality: Polyclonal

Regulatory Status: RUO

Target Species:

Human
Mouse

Applications:

Enzyme-Linked Immunosorbent Assay (ELISA)
Immunohistochemistry (IHC)
Western Blot (WB)

Storage:

SAMHD1 (phospho Thr592) antibody can be stored at 4˚C for three months and -20˚C, stable for up to one year.

Images

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<strong>Figure 1 Western Blot Validation in Human 293 Cells</strong><br>
Loading: 15 μg of lysates per lane.
Antibodies: SAMHD1 (phospho Thr592) 8005 (1 μg/mL), (1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
293 cells were transfected with
(A) empty expression vector,
(B) wild-type SAMHD1,
(C) SAMHD1 (mutation T592A) and
(D) SAMHD1 (mutation T592E)

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<strong>Figure 2 Mutant Validation in 293T Transfected Cells</strong><br>
Loading: 15 μg of lysates per lane.
Antibodies: SAMHD1 (phospho Thr592) 8005 (0.05 μg/mL), (1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
293 cells were transfected with (1) wild-type SAMHD1 or (2) SAMHD1 (mutation T592A).

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<strong>Figure 3 Immunohistochemistry Validation of SAMHD1 (phospho Thr592) in Human Brain Tissue </strong><br>
Immunohistochemical analysis of paraffin-embedded Human Brain Tissue using anti- SAMHD1 (phospho Thr592) antibody (8005) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4 ˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

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<strong>Figure 4 Immunofluorescence Validation of SAMHD1 (phospho Thr592) in Human Brain Tissue</strong><br>
Immunofluorescent analysis of 4% paraformaldehyde-fixed Human Brain Tissue labeling phospho SAMHD1 (phospho Thr592) with 8005 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).

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<strong>Figure 5 Mutant Validation of SAMHD1 (phospho Thr592) in human embryonic kidney (HEK) 293T cells (Antonucci et al., 2016) </strong><br>
WB analysis showed protein expression level of SAMHD1 (phospho Thr592) detected by anti- SAMHD1 (phospho Thr592) antibody (8005). SAMHD1 (phospho Thr592) expression was detected only in SAMHD1 WT, but neither in SAMHD1 T592A mutant nor the control group (empty vector).

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<strong>Figure 6 Mutant Validation of SAMHD1 (phospho Thr592) in primary monocyte-derived macrophages (MDM) from a healthy donor and human monocytic U937 cells (Antonucci et al., 2016) </strong><br>
The expression levels of SAMHD1 (phospho Thr592) detected by anti- SAMHD1 (phospho Thr592) antibody (8005) in SAMHD1 mutants (D137N & Q548A) of U937 cells are lower than those of SAMHD1 WT or MDM.

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<strong>Figure 7 Western Blot Validation of SAMHD1 (phospho Thr592) in herpes simplex viruses (HSV-1) infected human macrophages (Kueck et al., 2018) </strong><br>
HSV-1 infected macrophages were treated with aryl hydrocarbon receptor (AhR) agonist or antagonist. WB analysis showed protein expression level of SAMHD1 (phospho Thr592) detected by anti- SAMHD1 (phospho Thr592) antibody (8005). AhR agonist repressed SAMHD1 (phospho Thr592) expression as compared to the control group or AhR antagonist. Representative of 2 different donors.

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<strong>Figure 8 KD Validation of SAMHD1 (phospho Thr592) in human Macrophages (Mlcochova et al., 2017) </strong><br>
SAMHD1 (phospho Thr592) signal detected by anti- SAMHD1 (phospho Thr592) antibody (8005) in SAMHD1 knockdown cells were disrupted as compared to control.

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<strong>Figure 9 Western Blot Validation of SAMHD1 (phospho Thr592) in normal human fibroblasts (Tramentozzi et al., 2018) </strong><br>
WB analysis showed protein expression level of SAMHD1 (phospho Thr592) detected by anti- SAMHD1 (phospho Thr592) antibody (8005). SAMHD1 (phospho Thr592) expression was detected in S and G2/M phase of cell cycle but not in G1 phase.

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<strong>Figure 10 Regulated Expression Validation of SAMHD1 (phospho Thr592) in parental THP-1 cells (Tramentozzi et al., 2018) </strong><br>
WB analysis showed protein expression level of SAMHD1 (phospho Thr592) detected by anti- SAMHD1 (phospho Thr592) antibody (8005). SAMHD1 (phospho Thr592) expression was significantly decreased in S and G2/M phase with the treatment of lambda-phosphatase.

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<strong>Figure 11 Western Blot Validation of SAMHD1 (phospho Thr592) in Mouse SAMHD1 Transfected Cells (Wang et al., 2016) </strong><br>
WB analysis showed protein expression level of SAMHD1 (phospho Thr592) detected by anti-SAMHD1 (phospho Thr592) antibody (8005). Mouse SAMHD1 was detected in transfected cells with WT mSAMHD1, but not with mutants.

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<strong>Figure 12 Western Blot Validation of SAMHD1 (phospho Thr592) in Mouse Cells (Wang et al., 2016) </strong><br>
WB analysis showed protein expression level of SAMHD1 (phospho Thr592) detected by anti-SAMHD1 (phospho Thr592) antibody (8005). Mouse SAMHD1 was detected mouse MEF cells

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Documents

Further Information

Additional Names:

SAM domain and HD domain 1, DCIP, CHBL2, HDDC1, MOP-5, SBBI88

Application Note:

WB: 0.05-1 μg/mL; IHC: 2.5 μg/mL; IF: 20 μg/mL.

Antibody validated: Western Blot in human and mouse samples, Immunohistochemistry and Immunofluorescence in human samples. All other applications and species not yet tested.

Background:

The SAM domain and HD domain 1 (SAMHD1) protein is upregulated in response to viral infection and is thought to play a role in innate immunity (1). SAMHD1 blocks the infection of HIV-1 and SIVdeltaVpx before reverse transcription in macrophages and dendritic cells (2), and this restriction is regulated by phosphorylation of SAMHD1 (3). Mutations in this gene have been associated with Aicardi-Goutieres syndrome (1).

Background References:

Rice et al. Nat. Genet. 2009; 41:829-32.
Hrecka et al. Nature 2011; 474:654-7.
Welbourn et al. J. Virol. 2013; 87:11516-24.

Buffer:

SAMHD1 (phospho Thr592) antibody is supplied in PBS containing 0.02% sodium azide.

Concentration:

1 mg/mL

Conjugate:

Unconjugated

DISCLAIMER:

Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.

Immunogen:

Anti-SAMHD1 (phospho Thr592) antibody (8005) was raised against a phosphorylated peptide corresponding to 18 amino acids near the carboxy terminus of human Phospho-SAMHD1 surrounding T592.

ISOFORMS:

Human SAMHD1 (phospho Thr592) has 3 isoforms, including isoform 1 (626aa, 72kD), isoform 3 (556aa, 64kD), isoform 4 (591aa, 68kD). Mouse SAMHD1 has 2 isoforms, including (658aa, 76kD) and (651aa, 75kD). Rat SAMHD1 has only one isoform (620aa, 72kD). 8005 can detect human and mouse.

NCBI Gene ID #:

25939

NCBI Official Name:

SAM domain and HD domain 1

NCBI Official Symbol:

SAMHD1 (phospho Thr592)

NCBI Organism:

homo sapiens

Physical State:

Liquid

PREDICTED MOLECULAR WEIGHT:

Predicted: 72kD

Observed: 72 kD

Protein Accession #:

NP_056289

Protein GI Number:

38016914

Purification:

SAMHD1 (phospho Thr592) antibody is affinity chromatography purified via peptide column.

Research Area:

Immunology,Innate Immunity

SPECIFICITY:

Phospho-SAMHD1 antibody will only detect the phosphorylated form of SAMHD1.

Swissprot #:

Q9Y3Z3

User NOte:

Optimal dilutions for each application to be determined by the researcher.

VALIDATION:

Mutant validation (Figure 2, 5, 6 & 7): Anti-SAMHD1 (phospho Thr592) antibody (8005) specificity was further verified by SAMHD1 (phospho Thr592) mutant. SAMHD1 (phospho Thr592) signal in 293T cells (figure 2) or HEK293T cells (figure 5 & 7) or U937 cells (figure 6) transfected with SAMHD1 (phospho Thr592) mutant was disrupted in comparison with that in cells transfected with the wild-type.

KD validation (Figure 8): SAMHD1 (phospho Thr592) expression detected by anit-SAMHD1 (phospho Thr592) antibodies (8005) was disrupted in knockdown macrophages.

Regulated expression validation (Figure 10): SAMHD1 (phospho Thr592) expression detected by anit-SAMHD1 (phospho Thr592) antibodies (8005) was down-regulated by lambda-phosphatase treatment in S and G2 phase of cell cycle.

References

Antonucci et al. SAMHD1-mediated HIV-1 restriction in cells does not involvePMID: 27711056
Kueck et al. The aryl hydrocarbon receptor and interferon gamma generate antiviral states via transcriptional repression. Elife. 2018;7. PMID: 30132758
St Gelais et al. A Cyclin-Binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation. J Virol. 2018;92(6)PMID: 29321329
Tramentozzi et al. The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592. Cell Cycle. 2018;17(9):1102-1114. PMID: 30039733
Mlcochova et al. A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages. EMBO J. 2017;36(5):604-616.PMID: 28122869
Mlcochova et al. DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages. EMBO J. 2018;37(1):50-62. PMID: 29084722
Herrmann et al. The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation. Mob DNA. 2018;9:11. PMID: 29610582
Osei et al. USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1. J Virol. 2018;92(20). PMID: 30068654
St Gelais et al. A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability. J Biol Chem. 2016;291(51):26332-26342. PMID: 27815502
Wittmann et al. Phosphorylation of murine SAMHD1 regulates its antiretroviral activity. Retrovirology. 2015;12:103.PMID: 26667483
Feifei Wang. Comparison of Murine and Human SAMHD1?s Role in Retroviral Restriction and Cell Cycle Progression. PhD Thesis. The Ohio State University. 2015.
Alexandra Herrmann. The role of SAMHD1 in restriction and immune sensing of retroviruses and retroelements. PhD Thesis. The Friedrich Alexander University Erlangen-Nuremberg. 2018.