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: Primary antibodies used in the study and their resources were SAMHD1 (catalog number ab67820, Abcam), Tubulin (ab7291, Abcam), IKKα (2682, Cell Signaling), IKKα (61294S Cell Signaling), IKKβ (8943, Cell Signaling), Phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), IKKγ (2685, Cell Signaling), Phospho-IKKγ (Ser376) (2689, Cell Signaling), IκBα (4814, Cell Signaling), Phospho-IκBα (Ser32/36) (9246, Cell 12 J. Biol.

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: Primary antibodies used in the study and their resources were SAMHD1 (catalog number ab67820, Abcam), Tubulin (ab7291, Abcam), IKKα (2682, Cell Signaling), IKKα (61294S Cell Signaling), IKKβ (8943, Cell Signaling), Phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), IKKγ (2685, Cell Signaling), Phospho-IKKγ (Ser376) (2689, Cell Signaling), IκBα (4814, Cell Signaling), Phospho-IκBα (Ser32/36) (9246, Cell 12 J. Biol.

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: Primary antibodies used in the study and their resources were SAMHD1 (catalog number ab67820, Abcam), Tubulin (ab7291, Abcam), IKKα (2682, Cell Signaling), IKKα (61294S Cell Signaling), IKKβ (8943, Cell Signaling), Phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), IKKγ (2685, Cell Signaling), Phospho-IKKγ (Ser376) (2689, Cell Signaling), IκBα (4814, Cell Signaling), Phospho-IκBα (Ser32/36) (9246, Cell 12 J. Biol.

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: Primary antibodies used in the study and their resources were SAMHD1 (catalog number ab67820, Abcam), Tubulin (ab7291, Abcam), IKKα (2682, Cell Signaling), IKKα (61294S Cell Signaling), IKKβ (8943, Cell Signaling), Phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), IKKγ (2685, Cell Signaling), Phospho-IKKγ (Ser376) (2689, Cell Signaling), IκBα (4814, Cell Signaling), Phospho-IκBα (Ser32/36) (9246, Cell 12 J. Biol.

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

Journal: international Journal of Oncology

Article Title: Tautomycetin suppresses the TNFα/NF-κB pathway via inhibition of IKK activation

doi: 10.3892/ijo_00000091

Figure Lengend Snippet: Figure 4. Effects of OA and TC on IKK activation in 293-T cells. (A) 293-T cells were treated with vehicle (open circles), 100 nM OA (open triangles) or 5 μM TC (closed circles) for 5 h and then stimulated for the indicated periods with 40 ng/ml TNFα. IKK kinase activity was quantitated by dividing levels of radiolabeled-phospho-GST-IκBα (1-53) by levels of IKKß, determined by autoradiography using a Fluoro image analyzer and immunoblotting with an anti-IKKß antibody, respectively. IKK activity is expressed as fold activation relative to vehicle-treated sample at Time 0. (B) 293-T cells were treated with vehicle (Dose 0) or TC at the indicated dose for 5 h, stimulated for 10 min with 10 ng/ml TNFα and analyzed for IKK activity. IKK activity is calculated in a manner identical to that described in A. IKK activity is expressed as a percentage of that seen in the vehicle-treated control. Data are representative of three independent experiments.

Article Snippet: Specific antibodies to phospho-IKKα/ß (T23) [sc-21660], IKKα [sc-7606], IκBα [sc-371], IKKß [sc-7607], IKKγ [sc8256 and sc-8330], PP1C [sc-7482], TAK1 [sc-7967] and TRAF2 [sc-876] were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Activity Assay, Autoradiography, Western Blot, Control

Journal: international Journal of Oncology

Article Title: Tautomycetin suppresses the TNFα/NF-κB pathway via inhibition of IKK activation

doi: 10.3892/ijo_00000091

Figure Lengend Snippet: Figure 5. Effects of TC on IKKα and IKKß phosphorylation. 293-T cells (A and B) or HeLa and COS-7 cells (C) were treated with vehicle (control) or 5 μM TC for 5 h and then stimulated for the indicated periods with 10 ng/ml TNFα. Lysates were immunoprecipitated and blotted using the anti-IKKγ antibody. (A) Immunoblots with anti-phospho-IKKα/ß (S180/S181), anti- IKKß, anti-IKKα, anti-IκBα and anti-actin antibodies. Degradation of IκBα in lysates was seen following TNFα treatment. Actin served as an internal standard. (B) Immunoprecipitates were immunoblotted with anti-phospho- IKKα/ß (T23) and anti-IKKα antibodies. (C) Immunoprecipitates were immunoblotted with anti-phospho-IKKα/ß (S180/S181), anti-IKKß and anti-IKKα antibodies.

Article Snippet: Specific antibodies to phospho-IKKα/ß (T23) [sc-21660], IKKα [sc-7606], IκBα [sc-371], IKKß [sc-7607], IKKγ [sc8256 and sc-8330], PP1C [sc-7482], TAK1 [sc-7967] and TRAF2 [sc-876] were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Control, Immunoprecipitation, Western Blot

Journal: international Journal of Oncology

Article Title: Tautomycetin suppresses the TNFα/NF-κB pathway via inhibition of IKK activation

doi: 10.3892/ijo_00000091

Figure Lengend Snippet: Figure 7. PP1C associates with the IKK complex in 293-T cells. (A) 293-T cells were stimulated for the indicated periods with 10 ng/ml TNFα. Immunoblots of cell lysates and immunoprecipitates with either normal IgG or anti-IKKα were analyzed using anti-IKKα, anti-IKKß, anti-IKKγ, anti- PP1C, anti-IκBα and anti-actin antibodies. (B) 293-T cells were treated with vehicle or 5 μM TC for 5.5 h and then stimulated for the indicated periods with 10 ng/ml TNFα. Following immunoprecipitation with either normal IgG or anti-IKKα immunoblots were analyzed using anti-IKKα, anti-IKKß, anti-PP1C, anti-IκBα and anti-actin antibodies. In a separate experiment, we confirmed that the upper band of IκBα is a phosphorylated form using phospho antibody (data not shown).

Article Snippet: Specific antibodies to phospho-IKKα/ß (T23) [sc-21660], IKKα [sc-7606], IκBα [sc-371], IKKß [sc-7607], IKKγ [sc8256 and sc-8330], PP1C [sc-7482], TAK1 [sc-7967] and TRAF2 [sc-876] were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Short-Term Microgravity Influences Cell Adhesion in Human Breast Cancer Cells

doi: 10.3390/ijms20225730

Figure Lengend Snippet: List of the names, sources, companies, molecular weight and dilutions of all the antibodies that were used for Western blots.

Article Snippet: IKBKG , 38 , 1:500 , Origene #TA812460 , MS.

Techniques: Molecular Weight, Western Blot

Journal: Journal of Neurochemistry

Article Title: GABA B Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell‐Derived Sensory Neurons by Baclofen and α‐Conotoxin Vc1.1

doi: 10.1111/jnc.70004

Figure Lengend Snippet: Generation of H9 NGN2 human pluripotent stem cell (hPSC)‐derived induced sensory neurons (iSNs). (A) Schematic protocol for the differentiation of iSNs. Briefly, hPSC are differentiated to neural crest cells (NCCs, days 1–6), then enriched for the cell surface marker CD271 via magnetic cell sorting. CD271 + NCCs are plated for sensory neuron differentiation (days 6–27), with induction of NGN2 expression via application of doxycycline for 96 h (days 6–10). (A) (I–IV) Representative micrographs of H9 NGN2 through differentiation to iSNs; (I) hPSC colonies, scale bar = 500 μm; (II) NCC after 6 days differentiation, scale bar = 500 μm; (III) 96 h NGN2‐induced neurite outgrowth, scale bar = 200 μm; (IV) iSNs after 3 weeks differentiation and maturation, scale bar = 50 μm. (B–F). Immunocytochemistry staining for molecular markers through hPSC differentiation to iSNs. (B) H9 NGN2 hPSC stained for pluripotency transcription factors SOX2 (green) and OCT4 (red). (C) H9 NGN2 ‐derived NCC stained for cell surface receptor CD271 (green) and transcription factor SOX10 (red). (D) iSNs stained for BRN3A (green) and ISL1 (red) transcription factors. (E) iSNs stained for neural marker TUJ1 (green) and peripheral neuronal marker PRPH (red). (F) iSNs stained for neurogenesis transcription factors NGN1 (green) and NGN2 (red).

Article Snippet: Antibodies used for immunocytochemistry were those against SOX2 (AF2018; R&D Systems), OCT4 (sc‐5279 Santa Cruz Biotechnology, Dallas, TX, USA), CD271 (M‐1818‐100; Biosensis, Thebarton, SA, Australia), SOX10 (AF2684; R&D Systems, Minneapolis, MN, USA), BRN3A (MAB1585; Millipore, St. Louis, MO, USA), ISLET1 (ab20670; Abcam, Cambridge, UK), TUJ1 (MAB1637, Millipore), TUJ1 (ab18207, Abcam), Peripherin (ab4666, Abcam), NGN1 (MA5‐24900, Invitrogen), NGN2 (PA5‐78556, Invitrogen), GABBR1 (AP23115PU‐N; OriGene, Rockville, MD, USA), GABBR2 (ab75838, Abcam), Ca V 2.2 (KP10001, CALBIOCHEM), GIRK1 (APC‐005; Alomone Labs, Jerusalem, Israel), Na V 1.7 (ab65167, Abcam), Na V 1.8 (ab66743, Abcam), S100β (ab52642, Abcam), MAP2 (M4403, Sigma‐Aldrich), and PSD95 (#51–6900, Invitrogen).

Techniques: Derivative Assay, Marker, FACS, Expressing, Immunocytochemistry, Staining, Cell Surface Receptor Assay