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: 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