Journal: Carcinogenesis

Article Title: Cigarette smoke condensate activates nuclear transcription factor-kappaB through phosphorylation and degradation of IkappaB(alpha): correlation with induction of cyclooxygenase-2.

doi: 10.1093/carcin/23.9.1511

Figure Lengend Snippet: Fig. 4. (A) CSC induces degradation of IκBα. Two million U937 cells per ml were treated with either 0.1 nM TNF or 10 µg/ml CSC for different times. The cytoplasmic extracts were prepared and assayed for IκBα by western blot using IκBα-specific antibodies. (B) CSC activates IκBα kinase. Two million U937 cells per milliliter were treated with either 0.1 nM TNF or with 25 µg/ml CSC for different times. Thereafter the cytoplasmic extracts were prepared and assayed for IKK by the immunocomplex kinase assay (upper panel) and for IKKα (middle panel) and IKKβ (lower panel) protein by western blot analysis as described in Materials and methods.

Article Snippet: Monoclonal antibodies to IKKα and IKKβ were kindly provided by Imgenex (San Diego, CA) and antibodies against COX-2 were obtained from Transduction Laboratories (Lexington, KY).

Techniques: Western Blot, Kinase Assay

Journal: PLoS ONE

Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy

doi: 10.1371/journal.pone.0201858

Figure Lengend Snippet: (A ) RT-PCR of IL-8 mRNA in MDA-MB-231 cells pre-treated 12 h with control DMSO, Bay-117082 (10 or 40 μM), or SC-514 (10 or 40 μM), and incubated 24 h with 100 nM BZ. (B) Western analysis of IKKα, IKKβ, and IKKε in WCE of MDA-MB-231 cells transfected with control, IKKα, IKKβ, and IKKε siRNA, and treated with BZ (100 nM, 24 h). The bottom panel represents densitometric evaluation of IKKα, IKKβ, and IKKε protein levels shown in the top panel; the IKKs densities were normalized to actin, and expressed relative to cells transfected with control siRNA. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control, IKKα, IKKβ, or IKKε siRNA, and incubated 24 h with 100 nM BZ. (D) IL-8 release measured by ELISA in MDA-MB-231 cells transfected with control, IKKα, IKKβ, or IKKε siRNA and incubated 24 h with 100 nM BZ. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.

Article Snippet: Human IL-8 (sc-39631), IKKα (sc-29365), IKKβ (sc-35644), IKKε (sc-39056), and non-silencing (sc-37007) small interfering RNAs (siRNAs) were obtained from Santa Cruz Biotechnology.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Incubation, Western Blot, Transfection, Enzyme-linked Immunosorbent 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: 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: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: Effects of IKK1 and/or IKK2 KO on IL-3-induced expression of IEGs. ( A ) IKK1 and IKK2 protein levels in Ba/F3 parental cells (WT) and CRISPR/Cas9-mediated IKK1 KO, IKK2 KO, and IKK1/2 DKO cells. Whole-cell lysates were immunoblotted with the indicated Abs. A dagger denotes the detection of IKK2 by potential cross-reactivity of the anti-IKK1 Ab. ( B – D ) Relative levels of expression of c- fos ( B ), c- jun ( C ), and c- myc ( D ) mRNAs measured 20, 40, and 40 min, respectively, after IL-3 stimulation of Ba/F3 parental, IKK1 KO, IKK2 KO, and IKK1/2 DKO cells. * p < 0.05; n.s., not significant, as assessed by the Kruskal-Wallis test with the Steel-Dwass test. Each dot represents the result from independent experiments ( n = 5). Means are indicated by bars.

Article Snippet: The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [ ].

Techniques: Expressing, CRISPR

Journal: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: IL-3-induced IKK activation was not associated with the degradation of IκB-α followed by nuclear translocation of p65. ( A ) Phosphorylation of IKKs after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 for the indicated times, and whole-cell lysates were immunoblotted using the indicated Abs. Long and short exposures to detect phosphorylated IKK1 and IKK2 are shown. An arrowhead indicates the band corresponding to phosphorylated IKK1 at 5 min after IL-3 stimulation. A dagger denotes the detection of IKK2 by potential cross-reactivity of the anti-IKK1 Ab. ( B ) Phosphorylation of IKKs in IKK KO cells after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 parental (WT), IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were stimulated with IL-3 for 5 min. Immunoblot analysis was performed with whole-cell lysates using the indicated Abs. Arrowheads indicate the bands corresponding to phosphorylated IKK1. ( C ) Degradation of IκB-α by TNF-α but not by IL-3 stimulation. Following IL-3 deprivation, Ba/F3 cells were stimulated with IL-3 or TNF-α for the indicated durations, and whole-cell lysates were immunoblotted using the indicated Abs. ( D ) Induction of nuclear translocation of p65 by TNF-α but not by IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 or TNF-α for the indicated times and immunoblotted with Abs to the cytosol marker α-tubulin and the nuclear marker fibrillarin. Long and short exposures to detect p65 are shown.

Article Snippet: The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [ ].

Techniques: Activation Assay, Translocation Assay, Western Blot, Marker

Journal: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: IKK2-mediated activation of JNK regulates c- fos and c- jun expression. ( A ) Phosphorylation of JNK after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 for the indicated durations, and whole-cell lysates were subjected to immunoblot analysis using the indicated Abs. ( B – D ) Phosphorylation of JNK in IKK KO cells after IL-3 stimulation. Ba/F3 parental (WT), IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were stimulated with IL-3 after IL-3 deprivation for 6 h. Immunoblot analysis was performed with whole-cell lysates using the indicated Abs. ( E ) Expression levels of c- fos , c- jun , and c- myc mRNAs 20 and 40 min after IL-3 stimulation in the presence of JNK-IN-8 or DMSO. Ba/F3 cells were pre-incubated with 0.5 µM JNK-IN-8 or DMSO for 6 h and then stimulated with IL-3. * p < 0.05; ** p < 0.01; n.s., not significant, as assessed by Mann-Whitney U -tests, for differences between JNK-IN-8-treated and control cells at each time point. Each dot represents the result from independent experiments ( n = 5). Means are indicated by bars.

Article Snippet: The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [ ].

Techniques: Activation Assay, Expressing, Western Blot, Incubation, MANN-WHITNEY

Journal: EMBO Reports

Article Title: WNT signalling promotes NF-κB activation and drug resistance in KRAS-mutant colorectal cancer

doi: 10.1038/s44319-025-00588-1

Figure Lengend Snippet: ( A – F ) IHC staining of β-catenin or phospho-IKKα s176 in each genotype CRC sample. ( A ) KRAS WT (wild-type); ( B ) KRAS MT (mutation in position G12/G13); ( C ) KRAS WT plus low β-catenin; ( D ) KRAS MT plus low β-catenin; ( E ) KRAS WT plus high β-catenin; ( F ) KRAS MT plus high β-catenin expression in CRC samples. Scale bar 50 µm. ( G ) The graph shows the mean of expression of phospho-IKKα s176 in each different mutated human CRC, determined by IHC intensity values. Patients were grouped into four categories based on KRAS status and β-catenin expression. Both KRAS WT and β-catenin low ( n = 316); KRAS M and β-catenin low ( n = 164); KRAS WT and β-catenin high ( n = 59); Both KRAS M and β-catenin high ( n = 29). Each dot displays an individual sample. ( H , I ) Cell viability assay for T84 or SW620 colon cancer cells, showing the results of three experimental replicates, treatment with 0.1% DMSO, 50 nM trametinib or 10 µM JSH-23. The statistical tests used to calculate the P value are as follows: ( G ) one-way ANOVA; ( H , I ) 2-way ANOVA; NS P (>0.12), * P (0.033), ** P (0.002) and *** P (<0.001). All statistical data are summarised in Table . The error bar is a standard deviation (SD), with each point representing biological replicates.

Article Snippet: Sections were incubated in primary antibody β-catenin ((M3539, Dako, CA, USA, 1:600), IKKα (GWB-662250, Genway, CA, USA, 1:4000), IKKβ (ab32135, Abcam, Cambridge, UK, 1:200) and IKKα s176 (ab138426, Abcam, Cambridge, UK, 1:150) overnight at 4 o C. Sections were washed in tris-buffered saline (TBS), incubated in Impress secondary antibody (MP-7500, Vector Laboratories, CA, USA) for 2 h at room temperature.

Techniques: Immunohistochemistry, Mutagenesis, Expressing, Viability Assay, Standard Deviation

Journal: EMBO Reports

Article Title: WNT signalling promotes NF-κB activation and drug resistance in KRAS-mutant colorectal cancer

doi: 10.1038/s44319-025-00588-1

Figure Lengend Snippet: ( A – H ) Representative immunohistochemical (IHC) staining for IKKβ and IKKα in stage 2–3 colorectal cancer patient samples. ( A , E ) KRAS WT (wild-type) plus low β-catenin expression CRC samples; ( B , F ) KRAS MT (mutation in position G12/G13) plus low β-catenin expression CRC samples; ( C , G ) KRAS WT plus high β-catenin expression CRC samples; ( D , H ) KRAS MT plus high β-catenin expression CRC samples. The black arrow highlights the high signal area. Scale bar 50 µm. ( I , J ) The graph shows the mean of expression of IKKβ ( I ) or IKKα ( J ) in each different mutated human CRC, determined by IHC intensity values. Patients were grouped into four categories based on KRAS status and β-catenin expression. ( I ) Both KRAS WT and β-catenin low ( n = 311); KRAS M and β-catenin low ( n = 164); KRAS WT and β-catenin high ( n = 56); Both KRAS M and β-catenin high ( n = 28). ( J ) Both KRAS WT and β-catenin low ( n = 291); KRAS M and β-catenin low ( n = 158); KRAS WT and β-catenin high ( n = 57); Both KRAS M and β-catenin high ( n = 31). Each dot displays an individual sample. The statistical tests used to calculate the P value are as follows: ( I , J ) one-way ANOVA; NS P (>0.12), * P (0.033), ** P (0.002) and *** P (<0.001). All statistical data are summarised in Table . The error bar is a standard deviation (SD), with each point representing biological replicates. .

Article Snippet: Sections were incubated in primary antibody β-catenin ((M3539, Dako, CA, USA, 1:600), IKKα (GWB-662250, Genway, CA, USA, 1:4000), IKKβ (ab32135, Abcam, Cambridge, UK, 1:200) and IKKα s176 (ab138426, Abcam, Cambridge, UK, 1:150) overnight at 4 o C. Sections were washed in tris-buffered saline (TBS), incubated in Impress secondary antibody (MP-7500, Vector Laboratories, CA, USA) for 2 h at room temperature.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Mutagenesis, Standard Deviation

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