ikk2 protein  (MedChemExpress)

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 1. SAMHD1 suppresses IKKα- and IKKβ-mediated NF-κB activation in the absence or presence of TNF-α. HEK293T cells were cotransfected with 50 ng pN3-3 × FLAG-IKKα (A), or 25 ng pN3-3 × FLAG-IKKβ (B), or 50 ng pN3- 3 ×FLAG-IKKαand25ngpN3-3 ×FLAG-IKKβ (C),theincreasedamounts ofpRK- HA-SAMHD1,pNF-κB-luciferase(50 ng),and TK-renilla(10 ng).Anemptyvector wasusedtomaintain the sameamount ofplasmidDNA ineach transfection. At 24h post transfection, cellswere treatedwith TNF-α (10ng/ml) for 2 h andthen luciferase assays were performed. Results are expressed relative to empty vector, untreated cells, which are set to 1. The t test was used for statistical significance. *p < 0.05; **p < 0.01 (compared with vector control, lane 2 or 7 in each group). The expression levels of indicated proteins were detected by Western blot and GAPDH was a loading control. V, empty vector control.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Activation Assay, Luciferase, Transfection, Plasmid Preparation, Control, Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 5. SAMHD1 interacts with IKKα and IKKβ in cells. A, THP-1 control cells and PMA-differentiated THP-1 control cells were infected with SeV (MOI = 10) for indicated times or mock treated. Coimmunoprecipitation was performed with SAMHD1 antibody, Mouse IgG was used as a negative control. Input and immunoprecipitation (IP) samples were analyzed by Western blot. B, The relative levels of IKKα and IKKβ were quantified by densitometry analysis, and the IgG control was set as 1. The t test was used for statistical significance compared with IgG control. *p < 0.05. The data shown in B represent three independent experiments. C and D, HEK293T cells were cotransfected with plasmids encoding HA-SAMHD1 and indicated FLAG-IKKα (85 kDa), FLAG-IKKβ (87 kDa), or FLAG-IKKγ (48 kDa) separately. HEK293T cells were harvested after 48 h transfection. FLAG antibody (C) or HA antibody (D) was used for IP. The same amount of mouse IgG or rabbit IgG was used as a negative control. Input and IP samples were analyzed by immunoblotting (IB).

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Control, Infection, Negative Control, Immunoprecipitation, Western Blot, Transfection

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 6. Recombinant SAMHD1 interacts directly with purified rIKKα or IKKβ in vitro. A, His-SAMHD1 (5.6 μg) with or without His-IKKα (1 μg) was incubated with Dynabeads Protein A and SAMHD1 antibody (left panel). His-IKKα (5.6 μg) with or without His-SAMHD1 (1 μg) was incubated with Dynabeads Protein A and IKKα antibody (right panel). Input and IP samples were analyzed by Western blot. B, His-SAMHD1 (5.6 μg) with or without His-IKKβ (1 μg) was incubated with Dynabeads Protein A and SAMHD1 antibody (left panel). His-IKKβ (5.6 μg) with or without His-SAMHD1 (1 μg) was incubated with Dynabeads Protein A and IKKβ antibody (right panel). Input and immunoprecipitation (IP) samples were analyzed by Western blot.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Recombinant, In Vitro, Incubation, Western Blot, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 7. The HD domain of SAMHD1 is required for its interaction with IKKα and IKKβ. A, schematic diagrams of SAMHD1 wildtype (WT) and truncated mutants (M1, M2, M3, M4, M6, and M7). B and C, HEK293T cells were cotransfected with plasmids expressing FLAG-IKKα (B), FLAG-IKKβ (C), or empty vector, HA-SAMHD1 WT or SAMHD1 truncated mutants (M1, M2, M4, M6, and M7) for 48 h. An empty vector was used to maintain the same amount of plasmid DNA in transfection. FLAG antibody was used for immunoprecipitation (IP). Input and IP samples were analyzed by Western blot. D and E, HEK293T cells were cotransfected with plasmids expressing FLAG-IKKα (D), FLAG-IKKβ (E), or empty vector, and HA-SAMHD1 WT or HA-SAMHD1 HD domain (M3) for 48 h. HA antibody was used for IP. Input and IP samples were analyzed by Western blot. FLAG antibody and p-IKKα/β antibody were used for immunoblotting. HD, histidine aspartic domain; IgG LC, IgG light chain; M, molecular weight marker; SAM, sterile alpha motif.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Molecular Weight, Marker, Sterility

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 8. SAMHD1 C-terminal truncation suppresses IKKα- or IKKβ- mediated NF-κB activation. HEK293T cells were cotransfected with 50 ng of pN3-3 × FLAG-IKKα (A), or 25 ng of pN3-3 × FLAG-IKKβ (B), pRK-HA-SAMHD1 WT (200 ng or 400 ng, respectively) or pRK-HA-SAMHD1 M4 (200 ng or 400 ng, respectively), pNF-κB-luciferase (50 ng), and a plasmid expressing TK-renilla (10 ng). An empty vector was used to maintain the same amount of plasmid DNA in each transfection. At 24 h post transfection, cells were treated with TNF-α (10 ng/ml) for 2 h and then luciferase assays were performed. Results are expressed relative to empty vector, untreated cells, which are set to 1. The t test was used for statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001 (compared with vector control, lane 2 or 8 in each group). The expression levels of indicated proteins were detected by Western blot, and GAPDH was a loading control. V, empty vector control.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Expressing, Transfection, Control, Western Blot

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 9. The KD of IKKα and ULD of IKKβ are required for their interaction with SAMHD1. A, schematic diagrams of IKKα WT and truncated mutants (M1-M5). B, HEK293T cells were cotransfected with plasmids expressing HA-SAMHD1 or empty vector and FLAG-IKKα WT or FLAG-IKKα truncated mutants (M1 to M5) for 48 h. HA antibody was used for immunoprecipitation (IP). Input and IP samples were analyzed by Western blot. C, schematic diagrams of IKKβ WT and truncated mutants (M1-M5). D, HEK293T cells were cotransfected with HA-SAMHD1 or empty vector and FLAG-IKKβ WT or FLAG-IKKβ truncated mutants (M1 to M5) for 48 h. HA antibody was used for IP. Input and IP samples were analyzed by Western blot. KD, kinase domain; NBD, NEMO-binding domain; SDD, scaffold dimerization domain; ULD, ubiquitin-like domain.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Ubiquitin Proteomics

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 10. Exogenous SAMHD1 inhibits the interaction between TAK1 and IKKα or IKKβ. A and B, HEK293T cells were cotransfected with plasmids expressing FLAG-TAK1 and increasing amounts of HA-SAMHD1 and either Myc-IKKα (A) or Myc-IKKβ (B). Immunoprecipitation (IP) was performed using FLAG antibody. Input and IP samples were analyzed by Western blot.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal: The Journal of biological chemistry

Article Title: The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

doi: 10.1016/j.jbc.2023.104750

Figure Lengend Snippet: Figure 11. SAMHD1 inhibits phosphorylation of IKKα/β/γ and interacts with IKKα and IKKβ. SeV infection or inflammatory stimuli (LPS, TNF-α, or IL-1β) are recognized by corresponding receptors and induce phosphorylation of IKKα/β/γ and IκBα (indicated with a letter P). Cytoplasmic SAMHD1 interacts with the IKKα kinase domain and IKKβ ubiquitin-like domain and disrupts the interaction between TAK1 and IKKα or IKKβ to inhibit phosphorylation of IKKα/β/γ induced by inflammatory stimuli or SeV infection. The NF-κB inhibitory protein IκBα is phosphorylated by IKKα/β and degraded by the proteasome to activate subsequent NF-κB target gene transcription. Thus, SAMHD1 inhibits IKKα- and IKKβ-mediated NF-κB activation. Our findings revealed negative regulatory mechanisms by which SAMHD1 suppresses IκBα phosphorylation. Ub, ubiquitination. This figure was created with BioRender.com.

Article Snippet: SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate.

Techniques: Phospho-proteomics, Infection, Ubiquitin Proteomics, Activation Assay