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: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

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: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

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: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Staining, Control

Journal: Oncotarget

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

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A-F. Representative Western blots analysis of IKKα, P-p65, p65, EGFR, P-EGFR, p100/52, Maspin, c-Myc, E-cadherin and MMP-9 expression in Control, C-IKKα and N-IKKα tumors. Actin and GAPDH were used as loading controls. Western blot of protein extracts from 5 to 8 tumors derived from to 4 to 6 different mice of each genotype were performed. The identification of each tumor and mouse corresponding to every lane is provided in G. Bands of the different immunoblots were quantified by Quantity One software and Image Lab software and normalized with respect to Actin or GAPDH expression. P values were determined by Student's t -test and p values <0.05 (*) were considered significant; **p<0.01. H. Determination of VEGF-A mRNA relative levels in skin of Control, C-IKKα and N-IKKα transgenic mice by qRT-PCR analyses ( P <0,05).

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Western Blot, Expressing, Control, Derivative Assay, Software, Transgenic Assay, Quantitative RT-PCR

Journal: Oncotarget

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

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: Downregulation of E-cadherin expression in N-IKKα- and C-IKKα tumors. The CD31 staining (marker of endothelial cells) shows the presence of dilated and leaky blood vessels in the C-IKKα tumors, while those of Control and N-IKKα tumors are narrow and mature. Strong and delocalized suprabasal integrin-a6 staining is detected in N-IKKα tumors, while Control and C-IKKα tumors show Integrin-α6 basal expression. Reduced staining of Maspin in N-IKKα tumors. No difference in p52 and p65 expression was notice between tumors of the three genotypes.

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Staining, Marker, Control

Journal: Oncotarget

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

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A-C. Immunofluorescence with a Flag specific antibody showing the expression of the transgene in the nucleus of the HaCaT-N-IKKα cells (B) and in the cytoplasm of the HaCaT-C-IKKα cells (C). D. Representative western blot analyisis showing increased levels of IKKα in different pools of transfected HaCaT clones. Observe the increased MMP-9 and EGFR activation in the HaCaT-C-IKKα cells and the enhanced expression of c-Myc in the HaCaT-N-IKKα cells. E. Graphic representation of the densitometric analysis of western blots correponding to 6 pooled clones of HaCaT-C-IKKα cells, 3 pooled clones of HaCaT-N-IKKα cells and 3 pooled clones of HaCaT-Control cells. Student's t test was used for statistical analysis. (*p<0.05; ****p<0.0001).

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Immunofluorescence, Expressing, Western Blot, Transfection, Clone Assay, Activation Assay, Control

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a-c Human lung adenocarcinoma cell lines HCC827 ( a ), PC9 ( b ), and H1975 ( c ) stably overexpressing FLAG-tagged human DARPP-32 isoforms were lysed and subjected to immunoprecipitation using anti-IKKα antibodies. Immunoprecipitated lysates were separated in SDS-PAGE and immunoblotted with antibodies against IKKα, FLAG (that detects exogenously overexpressed DARPP-32), and α-tubulin (loading control).

Article Snippet: Human DARPP-32 isoforms purified from NSCLC cells were incubated with kinase-activated human IKKα protein (SignalChem, Cat no. C51-10G-10) in 50 μl reaction volume for in vitro kinase assays by following previously described methods .

Techniques: Stable Transfection, Immunoprecipitation, SDS Page, Control

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Human A549 cell lines stably overexpressing FLAG-tagged human DARPP-32 isoforms (DARPP-32 and t-DARPP) were lysed and subjected to immunoprecipitation using anti-FLAG antibody–conjugated agarose beads. Immunoprecipitated lysates were used to perform nonradioactive in vitro kinase assays following incubation with commercially available active IKKα protein. At the end, the reaction mixtures were subjected to immunoblotting using antibodies against DARPP-32 phosphorylated on Thr-34 or Thr-75 and total DARPP-32 protein. b , c Human HCC827, PC9, and H1975 lung adenocarcinoma cell lines retrovirally transduced with either FLAG-tagged human DARPP-32 ( b ) or t-DARPP ( c ) cDNA plasmids were lysed, immunoprecipitated, incubated with active IKKα protein, and subjected to western blotting using anti-phospho (Thr-34 or Thr-75) DARPP-32 and anti-DARPP-32 antibodies. Data from one experimental replicate are shown. The experiments were repeated three times independently; each circle in a bar represents one experiment. Error bars indicate SEM. * P < 0.05; ns not significant.

Article Snippet: Human DARPP-32 isoforms purified from NSCLC cells were incubated with kinase-activated human IKKα protein (SignalChem, Cat no. C51-10G-10) in 50 μl reaction volume for in vitro kinase assays by following previously described methods .

Techniques: Stable Transfection, Immunoprecipitation, In Vitro, Incubation, Western Blot, Transduction

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a , b Human lung cancer cells, HCC827 ( a ) and H1650 ( b ), transfected with GFP (control), constitutively active IKKα, or kinase-dead IKKα were lysed using 1× RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated with 4–20% SDS-PAGE and transferred to polyvinyl difluoride membranes. Antigen-coated membranes were incubated overnight with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). c , d Vehicle (DMSO)- or calyculin A-treated human HCC827 ( c ) and H1650 ( d ) cells were lysed with 1× RIPA buffer and subjected to immunoblotting using anti-phospho PP1α (Thr320), -total PP1α, -phospho ERK (Thr202/Tyr204), -total ERK, -DARPP-32, and -α-tubulin (loading control) antibodies. Chemiluminescence signals were detected after incubating membranes with HRP-tagged secondary antibodies. Representative images from one experiment are shown, but results were validated by performing three independent biological repeats. Bar graphs at the right show quantification of the results from the three western blotting experiments. Error bars indicate SEM. * P < 0.05; ns not significant.

Article Snippet: Human DARPP-32 isoforms purified from NSCLC cells were incubated with kinase-activated human IKKα protein (SignalChem, Cat no. C51-10G-10) in 50 μl reaction volume for in vitro kinase assays by following previously described methods .

Techniques: Transfection, Control, SDS Page, Incubation, Western Blot

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a – d Human NSCLC HCC827 ( a , c ) and H1650 ( b , d ) cells transduced with retrovirus designed to overexpress either wild-type ( a , b ) or mutant (T34A) DARPP-32 ( c , d ) were transfected with GFP (control), kinase-dead (KD), full-length (FL), and constitutively active (CA) IKKα cDNAs were lysed using 1× RIPA buffer supplemented with protease inhibitors only. Equal amounts of proteins (500 ng) were immunoprecipitated using anti-PP1α antibodies. Immunoprecipitated cell lysates were subjected to in vitro phosphatase assays following incubation with either PP1α substrate or histone H1 peptide (control). Released phosphates in each reaction tube were determined by using a phosphate detection reagent. In vitro phosphatase experiments were repeated three times independently. Bar graphs represent the mean ± SEM of the three repeats, with each circle in a bar representing an independent experiment. A value of P ≤ 0.05 was considered significant, ns not significant, one-way ANOVA followed by Dunnett’s test. e – h Immunoprecipitated HCC827 ( e , g ) and H1650 ( f , h ) cell lysates separated with 4–20% SDS-PAGE were subjected to western blotting using anti-PP1α antibodies. Input cell lysates were blotted with antibodies against IKKα, DARPP-32, and α-tubulin (loading control).

Article Snippet: Human DARPP-32 isoforms purified from NSCLC cells were incubated with kinase-activated human IKKα protein (SignalChem, Cat no. C51-10G-10) in 50 μl reaction volume for in vitro kinase assays by following previously described methods .

Techniques: Transduction, Mutagenesis, Transfection, Control, Immunoprecipitation, In Vitro, Incubation, SDS Page, Western Blot

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Representative images of HCC827, PC9, and H1650 cells transduced with lentivirus encoding either LacZ shRNA or IKKα shRNAs forming colonies on soft-agar cell culture dishes 1 to 2 weeks after plating. b Human NSCLC HCC827, PC9, and H1650 cells transduced with lentivirus designed to silence LacZ (control) or IKKα protein expression were subjected to soft-agar colony formation assays to determine anchorage-independent cell growth. ImageJ was used to count colonies on the cell culture dishes after 1 to 2 weeks of incubation, and the number of counted colonies was plotted. Each circle on a graph represents an independent experiment. Soft-agar colony formation experiments were repeated at least six times. Error bars indicate SEM ( n = 6). Scale bar 200 µm. A value of P ≤ 0.05 was considered significant, one-way ANOVA followed by Dunnett’s test. c , d Lysates from HCC827 ( c ) and PC9 ( d ) cells transduced with either LacZ shRNA or IKKα shRNA were subjected to immunoblotting with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). Bar graphs at the right show values obtained from the densitometric quantification of the results from three western blotting experiments. * P < 0.05; Two-tailed unpaired t -test.

Article Snippet: Human DARPP-32 isoforms purified from NSCLC cells were incubated with kinase-activated human IKKα protein (SignalChem, Cat no. C51-10G-10) in 50 μl reaction volume for in vitro kinase assays by following previously described methods .

Techniques: Transduction, shRNA, Cell Culture, Control, Expressing, Incubation, Western Blot, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Regulation of Th17 differentiation by IKKα-dependent and -independent phosphorylation of RORγt

doi: 10.4049/jimmunol.1700457

Figure Lengend Snippet: IKKα interacts with RORγt and regulates Th17 differentiation. (A) List of RORγt-associated kinases in Th17 cells, as identified by mass spectrometer. (B) IKKα-RORγt interaction in Th17 cells, as detected by westernblot analysis of immunoprecipitated RORγt. IgG is the control antibody used for immunoprecipitation (IP). (C) IKKα interaction with IL-17 promoter, as determined by ChIP assay. Enrichment is determined by signals obtained using anti-IKKα antibody relative to that with IgG control antibody. Hemoglobin gene (HBB) locus is used as a control. (D) Expression of IKKα and IKKβ in CD4+ T cells transduced with LMP retrovirus expressing indicated shRNA to knockdown IKKα or IKKβ, as determined by westernblot analysis. (E) Th17 differentiation of CD4+ T cells transduced with indicated shRNA shown in D, as determined by flow cytometric analysis of IL-17+ cells. (F) Quantification of the results shown in E. (G) Th17 differentiation of CD4+ T cells transduced with retrovirus expressing indicated proteins, as determined by flow cytometric analysis of IL-17+ cells. (H) Quantification of the results shown in G. (I) Expression of different IKKα/β in cells shown in G, as determined by westernblot analysis. Results in B, D, and I are the representatives of three independent experiments. Statistics are calculated based on three biological replica in C, F, and H.

Article Snippet: Hamster anti-RORγ Ab was purchased from BioLegend (San Diego, CA); rabbit anti-IKKα Ab and rabbit anti-IKKβ were purchased from Bethyl Laboratories (Montgomery, TX); mouse anti-SRC1 Ab, mouse anti-actin Ab, mouse anti-hemagglutinin (HA) Ab, and mouse anti-FLAG Ab were purchased from Sigma-Aldrich (St. Louis, MO); and rabbit anti-GFP Ab was purchased from Life Technologies (Carlsbad, CA).

Techniques: Mass Spectrometry, Immunoprecipitation, Control, Expressing, Transduction, shRNA, Knockdown

Journal: Scientific Reports

Article Title: Inhibition of Canonical NF-κB Signaling by a Small Molecule Targeting NEMO-Ubiquitin Interaction

doi: 10.1038/srep18934

Figure Lengend Snippet: ( a , b ) Effects of iNUB on TNFα and IL-1β-induced NF-κB signaling. MEFs were treated with 20 μM and 40 μM iNUB or DMSO 6 h before stimulation with TNFα ( a ) or IL-1β ( b ). NF-κB signaling was investigated by determining IκBα phosphorylation and degradation in Western Blot and NF-κB activation by EMSA. ( c , d ) iNUB does not affect JNK activation. MEFs were treated with DMSO or iNUB (40 μM) 6 h prior to stimulation with TNFα ( c ) or IL-1β ( d ). Phosphorylation of JNK and total JNK were analyzed by Western Blotting. ( e , f ) iNUB inhibits NF-κB target gene expression after TNFα, but not IL-1β stimulation. MEFs were treated with 20 μM and 40 μM iNUB or DMSO and stimulated with TNFα ( e ) or IL-1β ( f ). mRNA was isolated and NF-κB target genes were investigated by qRT-PCR. (n = 3; +/− SD) ( g ) iNUB induces apoptosis after TNFα stimulation. MEFs were stimulated for 24 h in the presence or absence of iNUB. Apoptosis was analyzed by AnnexinV staining and FACS. (n = 3; +/− SD) ( h ) iNUB impairs TNFα-induced IKK activation. MEFs were treated with 20 μM and 40 μM iNUB before TNFα stimulation. IKK activity after NEMO IP was determined by in vitro kinase assay using GST-IκBα 1–72 as the substrate. ( i , j ) iNUB does not directly inhibit IKK activity. The cellular IKK complex after TNFα stimulation and following NEMO IP ( i ) or recombinant IKKβ ( j ) were treated with iNUB before in vitro kinase reaction. IKKβ inhibitor (SC-514) served as positive control. ( k ) TNFα dependent recruitment of NEMO to ubiquitinated RIP1 via UBAN. ST-PD of StrepTagII-NEMO WT and D311N expressing MEF. Co-precipitation of ubiquitinated RIP1 in unstimulated or TNFα stimulated cells was performed and analyzed by Western Blot. ( l ) iNUB impairs recruitment of NEMO to ubiquitinated RIP1. ST-PD was performed with StrepTagII-NEMO WT after TNFα stimulation in the presence and absence of iNUB. Co-precipitated ubiquitinated RIP1 was analyzed by Western Blot.

Article Snippet: TNFα (50435; Biomol Germany); IL-1β (211-11B; PeproTech); antibodies: anti-IκBα (sc-371, Santa Cruz, USA), anti-p-IκBα (9246; Cell Signaling), anti-NEMO (FL-419, Santa Cruz), anti-β Actin (sc-1616, Santa Cruz), anti-IKKβ (05–535; Millipore), anti-IKKα (14A231; Imgenex), anti-JNK1/2 (9252; Cell Signaling), anti-p-JNK1/2 (9251; Cell Signaling), anti-Rip1 (3493; Cell Signaling) anti-FLAG M2 (F3165, Sigma); anti-mCD3 (553057, BD Biosciences); anti-hCD28 (553294, BD Biosciences); anti-IgG (307-005-003, Dianova).

Techniques: Western Blot, Activation Assay, Targeted Gene Expression, Isolation, Quantitative RT-PCR, Staining, Activity Assay, In Vitro, Kinase Assay, Recombinant, Positive Control, Expressing

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223-5p and -3p Cooperatively Suppress Necroptosis in Ischemic/Reperfused Hearts *

doi: 10.1074/jbc.M116.732735

Figure Lengend Snippet: A–C, representative Western blots and quantitative results of DR6 and TNFR1 protein expression in Ad.miR-223-infected rat cardiomyocytes (A), pre-miR-223 TG hearts (B), and pre-miR-223-null hearts (C) and their respective controls (n = 3 per group; *, p < 0.05 versus controls). D–F, protein levels of IKKα and NLRP3 were significantly reduced in Ad.miR-223-infected rat myocytes and pre-miR-223 TG hearts, whereas they were increased in pre-miR-223 KO hearts compared with the respective controls (n = 3 per group; *, p < 0.05 versus controls). Error bars represent mean ± S.D.

Article Snippet: The antibodies used in this study were as follows: rabbit anti-RIP1 (1:1000 dilution) (Santa Cruz Biotechnology, sc-7881 and Cell Signaling Technology, 3493), rabbit anti-RIP3 (1:1000 dilution) (Novus Biologicals, NBP1-77299), mouse anti-MLKL (1:1000 dilution) (Abcam Inc., ab194699), mouse anti-IKKα (1:500 dilution) (Novus Biologicals, NB100-56704), mouse anti-NLRP3 (1:330 dilution) (Adipogen AG-20B-0014), rabbit anti-DR6 (1:500 dilution) (Santa Cruz Biotechnology, sc-25772), and mouse anti-TNFR1 (1:250 dilution) (Santa Cruz Biotechnology, sc-8436). α-Actin (1:1000 dilution) (Sigma-Aldrich, A2172) was used as an internal control.

Techniques: Western Blot, Expressing, Infection

Journal: Journal of Neurotrauma

Article Title: Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood–Brain Barrier after Traumatic Brain Injury

doi: 10.1089/neu.2020.7229

Figure Lengend Snippet: Inhibitory effect of PRG4 on post-traumatic activation of NF-κB and ERK1/2 signaling cascades. rhPRG4 was injected i.v. at 1 h post-TBI at doses ranging from 1 to 5 mg/kg. NaCl solution (0.9%) was used as a vehicle. Extent of activation of the NF-κB signaling pathway in the injured cortex ( A ) was determined by assessing the level of phosphorylation (p) of IKKα/β on western blots. Extent of post-traumatic activation of ERK1/2 ( B ) was assessed based on the level of its phosphorylation. ( C ) Summary graphs illustrating the inhibitory effect of PRG4 on post-traumatic activation of IKKα/β and ERK1/2. PRG4 appears to inhibit activity of ERK1/2 with a higher efficacy than that of the NF-κB signaling cascade. Note that the lowest and highest rhPRG4 doses used (1 and 5 mg/kg) were the most effective in inhibiting post-traumatic activation of ERK1/2, especially at 24 and 48 h post-TBI. * p < 0.05; ** p < 0.01 for rhPRG4 versus vehicle ( n = 5–6 per group). ERK1/2, extracellular signal-regulated kinases 1/2; IKKα/β, IκB kinase α/β; i.v., intravenously; NF-κB, nuclear factor kappa B; p-ERK1/2, phosphorylated ERK1/2; p-IKKα/β, phosphorylated IKKα/β; PRG4, proteoglycan 4; rhPRG4, recombinant human PRG4; SDS, sodium dodecyl sulfate; TBI, traumatic brain injury.

Article Snippet: For western blotting, the following antibodies were used: rabbit monoclonal antihuman phosphorylated (p)-IKKα/β (IκB kinase α/β; Ser176/Ser180 [2697; clone 16A6, diluted 1:100; from Cell Signaling Technology, Danvers, MA]); rabbit polyclonal antihuman IKKα/β (orb157675; 1.25 μg/mL) from Biorbyt (Cambridge, UK); rabbit polyclonal antihuman p-ERK1/2 (extracellular signal-regulated kinases 1/2; Thr202/Tyr204; [9101; diluted 1:1000; from Cell Signaling Technology]); rabbit polyclonal antirat ERK1/2 (9102; diluted 1:2000; from Cell Signaling Technology); mouse monoclonal antirat cluster of differentiation 68 (CD68; MCA341R; clone ED1, 1 μg/mL; from Serotec, Oxford, UK); mouse monoclonal antimouse claudin 5 (CLDN5; 35–2500; clone 4C3C2, 0.25 μg/mL; from ThermoFisherScientific); mouse monoclonal antihuman β-actin (3700; clone 8H10D10, diluted 1:10,000; from Cell Signaling Technology); and goat polyclonal antirat albumin (55710; 0.4 μg/mL; from MP Biomedicals, Irvine, CA).

Techniques: Activation Assay, Injection, Western Blot, Activity Assay, Recombinant