Journal: International Journal of Medical Sciences

Article Title: Artemisia argyi -enhanced Mesenchymal Stem Cell Exosomes Alleviates Inflammation in C28/I2 Chondrocytes by inhibiting NF-κB

doi: 10.7150/ijms.126119

Figure Lengend Snippet: C28/I2 cells were exposed to H₂O₂ (100 μM, 2 h) followed by treatment with AA-enhanced WJSC-conditioned medium or exosomes. Cell viability, exosome uptake, NF-κB signaling proteins, cartilage-associated markers, and intracellular ROS levels were analyzed. (A) AA-enhanced conditioned medium at concentrations of 1%, 3%, 5%, 7%, and 9% for 24 hours significantly promoted C28/I2 cell proliferation compared to the control group. (B) After 2 hours of oxidative stress with H₂O₂, treatment with AA-enhanced conditioned medium restored cell viability in a dose-dependent manner. (C) Similarly, treatment with increasing concentrations of AA-enhanced WJSCs exosomes after H₂O₂ exposure enhanced cell viability. (D) Fluorescence microscopy confirmed cellular uptake of PKH67-labeled exosomes, with F-actin filaments stained in red (Scale bar = 20 μm). (E) Immunoblot analysis showed decreased expression of inflammation markers (p-IKKα/β, p-NF-κB, IκBα) and OA marker MMP13, with increased COL2A1 at higher concentrations of AA-enhanced conditioned medium (5%, 7%, 9%). (F) Similarly, AA-enhanced WJSCs exosomes (80 μg/mL) modulated inflammatory and OA markers. (G) mRNA expression of ADAMTS14, ADAMTS15, TNF-α, and IL-17A was normalized to β-actin using the 2-∆∆Ct method. (H) ROS generation was assessed by DCFDA staining. Fluorescence microscopy showed reduced ROS levels in cells treated with AA-enhanced exosomes (80 μg/mL) compared to the H₂O₂ group (Scale bar = 40 μm). (I) Quantification confirmed significant reduction in ROS levels, emphasizing the antioxidant effects of AA- conditioned vesicles. Data are presented as mean ± SD (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001 vs. untreated control group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. H₂O₂ treated group.

Article Snippet: The following antibodies were used in the current study: CD9 (#13174; Cell signaling technology, Danvers, MA, USA), CD63(Merck Millipore; Burlington, MA, USA), CD81(sc-166028; Santa Cruz Biotechnology, Dallas, TX, USA), Calnexin (#2679, Cell signaling), β-actin(sc-47778, Santa Cruz), p-IKKα/β (bs-3237R; Bioss Antibodies, Woburn, MA, USA), p-NF-κB p-p65 (#3033, Cell signaling), NF-κB p65(#8242, Cell signaling), p-IκB-α (#2859, Cell signaling), IκB-α (sc-1643, Santa Cruz), COL2A1 (GB11021; Servicebio, Wuhan, Hubei, China), MMP-13 (GTX100665; GeneTex, Irvine, CA, USA), Nanog (#4903, Cell Signaling), KLF4 (#4038, Cell signaling), CXCR4 (60042-1-Ig; Proteintech, San Diego, CA, USA), CXCR7 (bs-4897R, Bioss), CD44 (#3570, Cell signaling), CD90 (sc-53456, Santa Cruz).

Techniques: Control, Fluorescence, Microscopy, Labeling, Staining, Western Blot, Expressing, Marker

Journal: International Journal of Medical Sciences

Article Title: Artemisia argyi -enhanced Mesenchymal Stem Cell Exosomes Alleviates Inflammation in C28/I2 Chondrocytes by inhibiting NF-κB

doi: 10.7150/ijms.126119

Figure Lengend Snippet: Verification of AA-enhanced exosomes reducing H₂O₂-induced inflammation in C28/I2 cells and supporting cartilage homeostasis via inhibition of the NF-κB pathway using an NF-κB activator. C28/I2 cells were pretreated with H 2 O 2 for 2 hours and then co-treated with standard WJSCs exosomes or AA-enhanced WJSCs exosomes (80 μg/mL) with NF-κB activator (5μM) for 22 hours. (A) Western blot analysis reveals expression levels of key inflammation-related proteins, including p-IKKα/β, p-NF-κB, and IκBα, as well as OA markers MMP13 and COL2A1. (B) Translocation of p65 was determined using a NF-κB p65 antibody and an Alexa Fluor 488-conjugated anti-rabbit IgG antibody. Nuclei were counterstained with DAPI. Scale bar = 40 μm.

Article Snippet: The following antibodies were used in the current study: CD9 (#13174; Cell signaling technology, Danvers, MA, USA), CD63(Merck Millipore; Burlington, MA, USA), CD81(sc-166028; Santa Cruz Biotechnology, Dallas, TX, USA), Calnexin (#2679, Cell signaling), β-actin(sc-47778, Santa Cruz), p-IKKα/β (bs-3237R; Bioss Antibodies, Woburn, MA, USA), p-NF-κB p-p65 (#3033, Cell signaling), NF-κB p65(#8242, Cell signaling), p-IκB-α (#2859, Cell signaling), IκB-α (sc-1643, Santa Cruz), COL2A1 (GB11021; Servicebio, Wuhan, Hubei, China), MMP-13 (GTX100665; GeneTex, Irvine, CA, USA), Nanog (#4903, Cell Signaling), KLF4 (#4038, Cell signaling), CXCR4 (60042-1-Ig; Proteintech, San Diego, CA, USA), CXCR7 (bs-4897R, Bioss), CD44 (#3570, Cell signaling), CD90 (sc-53456, Santa Cruz).

Techniques: Inhibition, Western Blot, Expressing, Translocation Assay

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A. Recombinant DNA constructs employed to generate both transgenic mice lines. For C-IKKα mice generation, the nuclear localization signal (NLS) was removed from the sequence of the human IKKα cDNA employed. In the construct used for generation of the N-IKKα mice an extra NLS signal was added. WT IKKα; wild type IKKα. B. Western blot of total protein extracts showing IKKα expression in back skin of Control and C-and N-IKKα mice. Actin was used as a loading control. C. Representative example of the K5 staining in back skin section of Control mice. D-E. Expression of exogenous IKKα protein in back skin of 1-month-old mice. Immunostaining with the NB100-56704 anti-IKKα antibody is showed; similar results were obtained with the H00001147-M04 IKKα antibody (not shown). Note the cytoplasmic expression of the transgene in the C-IKKα mice (D). By contrast, it is located in the nuclei of cells in the N-IKKα mice (E). In both types of transgenic mice the exogenous IKKα is expressed in basal keratinocytes (bk), in the outer root sheath of hair follicles (ORS) and in cells surrounding the sebaceous glands (sb). F. Back skin section of Control mice. The NB100-56704 antibody used does not recognize the endogenous IKKα in immunohistochemical assays. G. Endogenous IKKα expression in control mice using the IKKα (sc-7182) antibody. Scale bar: (C) 70 μm; (D-G) 60 μm.

Article Snippet: Immunostaining was performed using antibodies against IKKα (NB100-56704) IKKβ (Novus Biologicals, Cambridge UK); IKKα (H00001147-M04) (Abnova, Taiwan); IKKα (sc-7182), P-IKKα/β (Ser 180/Ser 181)-R (sc-23470-R), Maspin, p65 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany); CD31, E- Cadherin, Integrin-α6 (BD Bioscience, NJ, USA); p52 (Abcam, Cambridge, UK).

Techniques: Recombinant, Construct, Transgenic Assay, Sequencing, Western Blot, Expressing, Control, Staining, Immunostaining, Immunohistochemical staining

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A. Western blot showing the increased expression of IKKα in transgenic mice. B-I. Immunohistochemistry showing the expression of the transgenic protein in N-IKKα and C-IKKa tumors. Staining with NB100-56704 antibody is shown. (B, C) Representative images showing the expression of transgenic IKKα in tumors and adjacent skin of N-IKKα/TgAC mice (B), and C-IKKα/TgAC animals (C). (D, E) Detail showing the nuclear (D) or cytoplasmic (E) localization of the transgenic IKKα in tumors. (F, G) Similar levels of expression of the transgenic IKKα in different N-IKKα tumors. By contrast variable levels of expression of the transgene are observed between different C-IKKα tumors (H, I). t: tumor; s: non-tumoral skin. Scale bar: (B, C) 100μm; (D, E) 80 μm; (F-I) 200 μm.

Article Snippet: Immunostaining was performed using antibodies against IKKα (NB100-56704) IKKβ (Novus Biologicals, Cambridge UK); IKKα (H00001147-M04) (Abnova, Taiwan); IKKα (sc-7182), P-IKKα/β (Ser 180/Ser 181)-R (sc-23470-R), Maspin, p65 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany); CD31, E- Cadherin, Integrin-α6 (BD Bioscience, NJ, USA); p52 (Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Transgenic Assay, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A, D, G. P-IKKα expression. P-IKKα/β (Ser 180/Ser 181) antibody is used. B, E, H. Specific staining of human IKKα-using the NB100-56704 antibody. C, F, I. Staining with the sc-7182 antibody that recognizes both human and mouse IKKα. Observe that as expected, in the N-IKKα tumors the signal of this antibody is detected both in cytoplasmic and nuclear localization; by contrast, in the Control tumors the endogenous IKKα is mainly observed in the cytosolic compartment, although some nuclear staining is also observed. In the C-IKKα tumors little nuclear staining is observed. Scale bar: 70μm.

Article Snippet: Immunostaining was performed using antibodies against IKKα (NB100-56704) IKKβ (Novus Biologicals, Cambridge UK); IKKα (H00001147-M04) (Abnova, Taiwan); IKKα (sc-7182), P-IKKα/β (Ser 180/Ser 181)-R (sc-23470-R), Maspin, p65 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany); CD31, E- Cadherin, Integrin-α6 (BD Bioscience, NJ, USA); p52 (Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Control

Journal: Journal of Clinical Investigation

Article Title: Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

doi: 10.1172/jci30556

Figure Lengend Snippet: Figure 1 NF-κB activity in dystrophic muscle is localized to both muscle and immune cells. (A–C) Muscle nuclear extracts from 5-week-old WT C57BL/10 and 3- and 5-week-old mdx mice (A), 5-week-old WT and mdx mice (B), or 7-week-old WT, mdx, and DKO mice (C) were used in EMSA for NF-κB and Oct-1. Supershift assays were performed on mdx muscle extracts using antibodies against p65 and p50. Arrowheads denote shifted subunits. (D) IKK assays performed with IκBα WT and mutant (double serine to threonine [SS/TT]) or p65 WT and mutant (serine to alanine [S/A]) substrates using gastrocnemius muscle lysates from 7-week-old WT or mdx mice. Immunoprecipitates were probed for IKKγ as a loading control. Western blots are shown for p-IKK, p-IκBα, IκBα, p-p65, and p65. GST, glutathione-S-transferase. (E) Gastrocnemius muscles from 7-week-old WT or mdx mice were immunostained for p-p65. Scale bars: 50 μm. Black arrowheads denote immune cells, and blue arrowheads indicate regenerating fibers. (F) Muscles from either 4- or 7-week-old mdx mice were double stained with p-p65 (green) and CD68 (red) or p-p65 and E-MyHC (red), respectively. Scale bars: 20 μm. (G) H&E staining and p-p65 immunohistochemistry were performed on muscle biopsies from healthy controls and age-matched DMD patients (n = 4). Scale bar: 50 μm.

Article Snippet: Muscle extracts for Western blot analysis were prepared as previously described (52) and probed using antibodies against p-p65, p-IκB, pIKK (1:500; Cell Signaling Technology), IκBα (1:500; Santa Cruz Biotechnology Inc.), IKKα (1:500; Imgenex), IKKβ 900 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 4 April 2007 (1:500; Cell Signaling Technology), IKKγ (1:500; Santa Cruz Biotechnology Inc.) and p65 (1:10,000; Rockland).

Techniques: Activity Assay, Mutagenesis, Control, Western Blot, Muscles, Staining, Immunohistochemistry

Journal: Journal of Clinical Investigation

Article Title: Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

doi: 10.1172/jci30556

Figure Lengend Snippet: Figure 6 IKKβ deletion in muscle cells promotes regeneration. Muscles harvested from 4-week- (A–F) or 12-week-old (F only) mdx;IKKβF/F and mdx;IKKβF/F;MLC-Cre mice were used for protein analysis (A and C) or histology (B and D). (A) Western blots probing for IKK subunits. (B) Muscles were immunostained for p-p65 expression. (C) Western blots probing for p-p65, p65, and IκBα. (D) H&E staining of mdx;IKKβF/F and mdx;IKKβF/F;MLC-Cre muscles. (E) Muscles used in D were stained for quantitation of E-MyHC–positive stained fibers or stained with H&E for counting of CLN. *P < 0.05. Scale bars: 15 μm (B) and 50 μm (D). Graphs are plotted as mean ± SEM. (F) Mean fiber distribution in mdx;IKKβF/F and mdx;IKKβF/F;MLC-Cre mice was determined from a minimum of 3,000 fibers from randomly chosen fields, obtained from multiple muscle sections from a minimum of 4 mice per group.

Article Snippet: Muscle extracts for Western blot analysis were prepared as previously described (52) and probed using antibodies against p-p65, p-IκB, pIKK (1:500; Cell Signaling Technology), IκBα (1:500; Santa Cruz Biotechnology Inc.), IKKα (1:500; Imgenex), IKKβ 900 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 4 April 2007 (1:500; Cell Signaling Technology), IKKγ (1:500; Santa Cruz Biotechnology Inc.) and p65 (1:10,000; Rockland).

Techniques: Muscles, Western Blot, Expressing, Staining, Quantitation Assay

Journal: Journal of Clinical Investigation

Article Title: Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

doi: 10.1172/jci30556

Figure Lengend Snippet: Figure 8 Pharmacological inhibition of IKK rescues the histopathology and function of dystrophic muscle. (A) Amino acid sequence of WT or mutant (mut) NBD peptides. Underlined amino acids indicate changes from WT to mutant forms. (B) Soleus muscles from WT or mutant NBD–treated mice were stained with F4/80, and macrophages were quantitated. Scale bar: 300 μm. (C) Gastrocnemius muscles harvested from mdx mice treated for 4 weeks were sectioned and stained with either p-p65 (C) or H&E (E). Scale bars: 20 μm (C and E). (D) Lysates from mice were used for Western blots probing for p-p65, p65, and IκBα. (F) RNA was isolated from similar muscles as used for D including C57BL/10 and mdx controls, and real-time PCR was performed for lysozyme (n = 4). (G) Regeneration potential was measured by quantitating fibers with centronucleation and positive E-MyHC staining from mdx mice treated with saline or NBD peptides. (H) Force generation assessed by measur- ing active developed force comparing diaphragm muscles from mice treated with either WT or mutant NBD peptide for 4 weeks. Quantitative data are plotted as mean ± SEM from 3 independent experiments. *P < 0.05.

Article Snippet: Muscle extracts for Western blot analysis were prepared as previously described (52) and probed using antibodies against p-p65, p-IκB, pIKK (1:500; Cell Signaling Technology), IκBα (1:500; Santa Cruz Biotechnology Inc.), IKKα (1:500; Imgenex), IKKβ 900 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 4 April 2007 (1:500; Cell Signaling Technology), IKKγ (1:500; Santa Cruz Biotechnology Inc.) and p65 (1:10,000; Rockland).

Techniques: Inhibition, Histopathology, Sequencing, Mutagenesis, Muscles, Staining, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Saline

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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 2. SAMHD1 inhibits phosphorylation of IKKα/β/γ induced by LPS treatment. A and C, THP-1 control (Ctrl) cells and THP-1 SAMHD1 knockout (KO) cells (A) or PMA-differentiated THP-1 ctrl cells and PMA-differentiated THP-1 SAMHD1 KO cells (C) were treated with LPS (100 ng/ml) for 15 min to 6 h or mock treated. The cell lysates were harvested at each time point and endogenous SAMHD1, IKKα/β/γ, p- IKKα/β, p-IKKγ, IκBα, p- IκBα, and tubulin were detected by Western blot. Tubulin was a loading control. B and D, The relative p-IKKα/β, p-IKKγ, and p-IκBα levels were quantified by densitometry analysis. Relative p-IKKα/β, p-IKKγ, and p-IκBα levels were normalized to tubulin to avoid the difference of total protein expression levels due to different treatments. The results were presented as means ± SD. Levels of each phosphoprotein are expressed relative to THP-1 control cells without LPS treatment, which were set to 1. The t test was used for statistical significance compared with THP-1 control cells. *p < 0.05; **p < 0.01. The data shown in B and D represent three independent experiments.

Article Snippet: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

Techniques: Phospho-proteomics, Control, Knock-Out, Western Blot, Expressing

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 3. SAMHD1 inhibits phosphorylation of IKKα/β/γ induced by SeV infection. A and C, THP-1 control cells and THP-1 SAMHD1 KO cells (A) or PMA- differentiated THP-1 control cells and PMA-differentiated SAMHD1 KO cells (C) were infected with SeV (multiplicity of infection [MOI] of 10) for 1 to 8 h or mock treated. The expression levels of SAMHD1, IKKα/β/γ, p- IKKα/β, p-IKKγ, IκBα, p- IκBα, SeV nucleoprotein (NP) protein, and tubulin were measured by Western blot. Tubulin was used as a loading control. B and D, The relative p-KKα/β, p-IKKγ, and p-IκBα levels were quantified by densitometry analysis. Relative p-KKα/β, p-IKKγ, and p-IκBα levels were normalized to tubulin. Levels of each phosphoprotein are expressed relative to THP-1 control cells without SeV infection which were set to 1. The t test was used for statistical significance compared with the THP-1 control cells. *p < 0.05; **p < 0.01. The data shown in B and D represent three independent experiments.

Article Snippet: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

Techniques: Phospho-proteomics, Infection, 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 4. SAMHD1 reconstitution inhibits phosphorylation of IKKα/β/γ induced by SeV infection. A and C, (A) THP-1 control cells, THP-1 SAMHD1 KO cells, THP-1 Lvx cells, and THP-1 SAMHD1 KI cells were infected with SeV (MOI = 10) for 4 h or mock treated. C, THP-1 control cells, THP-1 SAMHD1 KO cells, THP-1 Lvx cells, and THP-1 SAMHD1 KI cells were differentiated by PMA (30 ng/ml) for 48 h and then infected with SeV (MOI = 10) for 4 h or mock treated. The expression levels of SAMHD1, IKKα/β/γ, p- IKKα/β, p-IKKγ, IκBα, p-IκBα, SeV nucleoprotein (NP) protein, and tubulin were measured by Western blot. Tubulin was used as a loading control. B and D, The relative p-IKKα/β, p-IKKγ, and p-IκBα levels were quantified by densitometry analysis. The results were presented as means ± SD. Levels of each phosphoprotein are expressed relative to control cells infected with SeV for 4 h, which were set to 1. The t test was used for statistical significance. *p < 0.05; **p < 0.01. The data shown in B and D represent three independent experiments.

Article Snippet: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

Techniques: Phospho-proteomics, Infection, 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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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: Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

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