Journal:

Article Title: Activation of NF-kB by TMPRSS2/ERG fusion isoforms through Toll-like receptor-4

doi: 10.1158/0008-5472.CAN-10-2210

Figure Lengend Snippet: A. Western blots of extracts of PNT1a cells expressing Type III+72, Type VI+72 or vector control with anti-p65, anti-p65 phospho-Ser536 or β-actin antibodies (left); Similar Western blots oh VCaP cells expressing a fusion gene specific shRNA or vector controls (right). B. Western blot of PNT1a and multiple PCa cell lines with anti-p65, anti-p65 phospho-Ser536 or β-actin antibodies. C and D. Cos7 cells were transfected with NF-κB p65 along with Type III+72 or VI+72 fusion gene expression constructs or control vector (pCMV-Tag2B). TRITC-conjugated goat anti-rabbit antibody was used to detect total p65 (C) or phospho-Ser536 p65 (D) after incubation with anti-p65 or anti-p65 Ser536 antibody. Total positive nuclei per 10 high power fields (200×) is shown as mean +/− standard deviation of 10 fields. Statistically significant differences from controls by t-test are indicated by asterisks (p<.01).

Article Snippet: Immunohistochemistry Immunohistochemistry of VCaP orthotopic tumors was performed using a rabbit polyclonal p65 anti-phospho-Ser536 from Abgent, Inc (AP3178a) as described previously ( 14 ).

Techniques: Western Blot, Expressing, Plasmid Preparation, shRNA, Transfection, Construct, Incubation, Standard Deviation

Journal:

Article Title: Activation of NF-kB by TMPRSS2/ERG fusion isoforms through Toll-like receptor-4

doi: 10.1158/0008-5472.CAN-10-2210

Figure Lengend Snippet: A. Immunohistochemistry using anti-p65 Ser536 antibody was performed using a prostate cancer tissue microarray was performed and quantiated using a 10 point quantitation scale as described in Materials and Methods. Examples of staining with staining indices of 9, 6, 3 and 0 are shown. Original maginification 400×, dash equals 200 microns. B. Kaplan-Meier plot of recurrence free survival following radical prostatectomy of cancers with no staining with anti-p65 Ser536 antibody versus cancers with staining. C. Association of ERG expression with 65 phospho-Ser536 IHC. Cases with the indicated staining indices for p65 phospho-Ser536 by IHC 0, 1–3 (low), 4–6 (moderate) and 7–9 (high) were scored for ERG expression by IHC and the fraction of cases with ERG expression, which is highly correlated with expression of the T/E fusion gene was determined. The distribution of ERG positive cases between groups was significantly different than chance (p<.0001, Chi-square)

Article Snippet: Immunohistochemistry Immunohistochemistry of VCaP orthotopic tumors was performed using a rabbit polyclonal p65 anti-phospho-Ser536 from Abgent, Inc (AP3178a) as described previously ( 14 ).

Techniques: Immunohistochemistry, Microarray, Quantitation Assay, Staining, Expressing

Journal:

Article Title: Activation of NF-kB by TMPRSS2/ERG fusion isoforms through Toll-like receptor-4

doi: 10.1158/0008-5472.CAN-10-2210

Figure Lengend Snippet: A. Expression of TLR4 as determined by quantitative RT-PCR in PNT1a cells stably expressing the VI+72 fusion gene isoform transiently transfected with two plasmids expressing shRNas targeting TLR4 or control plasmid. B. Western blot of protein extracts on transiently transfected PNT1a cells described above using anti-p65 phospho-Ser536 antibody. β-actin is a loading control.

Article Snippet: Immunohistochemistry Immunohistochemistry of VCaP orthotopic tumors was performed using a rabbit polyclonal p65 anti-phospho-Ser536 from Abgent, Inc (AP3178a) as described previously ( 14 ).

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Western Blot

Journal: Neural Regeneration Research

Article Title: Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons ★

doi: 10.3969/j.issn.1673-5374.2013.16.003

Figure Lengend Snippet: Effect of lipopolysaccharide (LPS) on the expression of nuclear factor-kappa B (NF-κB) p65 and phospho-inhibitor of kappa B α (p-IκBα) protein at 0, 0.5, 2, 6 and 12 hours after LPS (10 μg/mL) stimulation determined by western blot analysis. (A) NF-κB p65 protein level in the nuclei of hippocampal neurons was detected by western blot analysis. Histone was used as an internal control in the nuclei. (B) p-IκBα protein level in the cytosol of each group was detected by western blot analysis. β-actin was used as an internal control. (C) Quantification of NF-κB p65 and p-IκBα protein expression. The mean densitometry values of NF-κB p65/histone and p-IκBα/β-actin in various groups were analyzed and expressed as bar charts to represent the relative expression of NF-κB p65 protein in the nuclei and p-IκBα protein in cytosol. a P < 0.01, vs . control group (0 h). Data are expressed as mean ± SD and n = 3 per group. One-way analysis of variance and comparisons between two groups were made with the least significant difference and Student-Newman-Keuls post-hoc test. h: Hour.

Article Snippet: Each membrane was incubated in fresh blocking buffer (containing 5% nonfat dry milk) at room temperature for 30 minutes, and was then incubated with rabbit anti-Toll-like receptor 4 polyclonal antibody (1:200; Santa Cruz Biotechnology), mouse anti-β-actin monoclonal antibody (1:2 000; Santa Cruz Biotechnology), mouse anti-histone H1 monoclonal antibody (1:500; Millipore, Billerica, MA, USA), rabbit anti-nuclear factor-kappa B p65 polyclonal antibody (1:1 000; Kangchen BIO-TECH, Shanghai, China) and mouse anti-phospho-inhibitor of kappa B α monoclonal antibody (1:1 000, Kangchen BIO-TECH) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons ★

doi: 10.3969/j.issn.1673-5374.2013.16.003

Figure Lengend Snippet: Effect of Toll-like receptor 4 (TLR4) antibody on the expression of nuclear factor-kappa B (NF-κB) p65, phospho-inhibitor of kappa B α (p-IκBα) and the production of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). (A) Cells were pretreated with TLR4 antibody (10 μg/mL) for 1 hour and were then stimulated with lipopolysaccharide (LPS; 10 μg/mL) for 2 hours. NF-κB p65 protein (65 kDa) levels in the nucleus of hippocampal neurons of different groups were detected by western blot analysis. Histone (33 kDa) was used as an internal control in the nuclei. (B) p-IκBα protein (41 kDa) level in the cytosol of each group was detected by western blot analysis. β-actin (43 kDa) was used as an internal control. (C) Relative levels of NF-κB p65 in the nucleus and p-IκBα in the cytosol. The mean densitometry values of NF-κB p65/histone and p-IκBα/β-actin in various groups were analyzed and expressed as bar charts to represent the relative expression of NF-κB p65 protein in the nucleus and p-IκBα protein in the cytosol. a P < 0.01, vs . control group; b P < 0.05, vs . LPS group. (D) Effect of TLR4 antibody on the concentration of IL-1β and TNF-α in culture supernatant. Cells were pretreated with TLR4 antibody (10 μg/mL) for 1 hour and were then stimulated with LPS (10 μg/mL) for 24 hours. The concentration of IL-1β and TNF-α in culture supernatant was detected by enzyme-linked immunosorbent assay. a P < 0.01, vs . control group; b P < 0.01, vs . LPS group. (C–D) Data are expressed as mean ± SD and n = 3 per group. One-way analysis of variance and comparisons between two groups were made with the least significant difference and Student-Newman-Keuls post-hoc test.

Article Snippet: Each membrane was incubated in fresh blocking buffer (containing 5% nonfat dry milk) at room temperature for 30 minutes, and was then incubated with rabbit anti-Toll-like receptor 4 polyclonal antibody (1:200; Santa Cruz Biotechnology), mouse anti-β-actin monoclonal antibody (1:2 000; Santa Cruz Biotechnology), mouse anti-histone H1 monoclonal antibody (1:500; Millipore, Billerica, MA, USA), rabbit anti-nuclear factor-kappa B p65 polyclonal antibody (1:1 000; Kangchen BIO-TECH, Shanghai, China) and mouse anti-phospho-inhibitor of kappa B α monoclonal antibody (1:1 000, Kangchen BIO-TECH) at 4°C overnight.

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Prothymosin α overexpression contributes to the development of pulmonary emphysema

doi: 10.1038/ncomms2906

Figure Lengend Snippet: ( a ) Detection of acetylated NF-κB p65 (Lys310). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviruses expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression vector (pCMVb p300 HA delta33) or a control vector (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression in the transfected cells were verified. ( b ) Reporter assay for NF-κB transactivation activity. 293T cells that had been transduced with lentiviruses expressing ProT-IRES-GFP or GFP alone (left), or ProT or GFP shRNA (right) were cotransfected with pNF-κB-Luc and pTK-Renilla reporter plasmids. Total cell lysates were harvested 48 h later, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light activity. Values shown are the mean±s.e.m. ( n =4 for left and n =5 for right; Student’s t -test). ( c ) NF-κB and ProT binding assay. Cell lysates from 293T cells transduced with lentiviruses expressing GFP-tagged ProT or GFP alone were immuoprecipitated by anti-GFP monoclonal antibody followed by immunoblotting with anti-NF-κB p65 and anti-GFP antibody. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of NF-κB and ProT by immunoblotting. ( d ) Analysis of the effect of ProT on NF-κB and HDAC3 binding. 293T cells that had been transfected with an NF-κB p65 expression vector (pcDNA3-cFlag-RelA) or a control vector (pCMV-Tag2B) (left) or HDAC3 expression vector (pcDNA3.1-HDAC3-Flag) or a control vector (pCMV-Tag2B) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. After 24 h, total cell lysates were immuoprecipitated by Flag-M2 beads followed by immunoblotting with the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated by immunoblotting with indicated proteins. ( e ) p300 and NF-κB binding assay. 293T cells that had been transduced with lentiviruses expressing ProT shRNA-2 or luciferase shRNA as the control were transfected with the pCMVb p300 HA delta33 and pcDNA3-cFlag-RelA. Total cell lysates were immunoprecipitated by anti-HA antibody followed by immunoblotting with the indicated proteins. Results are representative of three independent experiments. ( f ) Proposed model for ProT-mediated NF-κB hyperacetylation via HDAC3/NF-κB dissociation and p300/NF-κB association.

Article Snippet: The primary antibodies used included monoclonal antibodies against ProT (clone 2F11, ascites fluid) and polyclonal antibodies against HDAC1 (1:100, Santa Cruz), HDAC2 (1:100, Santa Cruz), HDAC3 (1:100, Santa Cruz), acetylated-lysine (1:800, Cell Signaling), acetylated-Lys310 NF-κB p65 (1:100, Genetex), MMP2 (1:50, Santa Cruz) and MMP9 (1:50, Santa Cruz).

Techniques: Transfection, Transduction, Expressing, shRNA, Plasmid Preparation, Control, Western Blot, Reporter Assay, Activity Assay, Luciferase, Binding Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: Prothymosin α overexpression contributes to the development of pulmonary emphysema

doi: 10.1038/ncomms2906

Figure Lengend Snippet: ( a – c ) Immunohistochemical detection of acetyl-NF-κB p65 in lung tissues from 4-day-old ProT HZ, HET and NT mice ( a ), from patients with emphysema of varying severity and non-COPD individuals ( b ) and from FVB mice that had been intratracheally injected with lentiviral vectors expressing ProT or luciferase (Luc) shRNA followed by intraperitoneal injections of CSE or saline twice a week for 6 weeks ( c ). Note that positive nuclear staining for acetyl-NF-κB p65 in the lung epithelium of ProT transgenic mice was observed ( a ) and that higher levels of acetyl-NF-κB p65 accumulation were detected in patients with more severe emphysema ( b ). The boxed areas in upper panels (original magnification × 400; Scale bar=20 μm) are magnified and shown in lower panels. ( d , e ) Immunofluorescence microscopy. Lung tissues from ProT HET and NT mice that had been injected intraperitoneally with CSE or saline twice a week for 6 weeks ( d ) and mouse lung epithelial MLE 12 cells that had been transduced with ProT and GFP by lentiviral vectors Lenti-ProT and Lenti-GFP, respectively, and then treated with CSE or saline ( e ) were incubated with mouse monoclonal antibody against ProT and rabbit polyclonal antibody against NF-κB p65 or acetyl-NF-κB p65 (Lys310) followed by Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Nuclei were counterstained with DAPI. Fluorescence signals for ProT (red), NF-κB p65 (green), acetyl-NF-κB p65 (green) and the nucleus (blue) were examined by fluorescence microscopy. Scale bar=10 μm; original magnification × 400. Results are representative of three independent experiments.

Article Snippet: The primary antibodies used included monoclonal antibodies against ProT (clone 2F11, ascites fluid) and polyclonal antibodies against HDAC1 (1:100, Santa Cruz), HDAC2 (1:100, Santa Cruz), HDAC3 (1:100, Santa Cruz), acetylated-lysine (1:800, Cell Signaling), acetylated-Lys310 NF-κB p65 (1:100, Genetex), MMP2 (1:50, Santa Cruz) and MMP9 (1:50, Santa Cruz).

Techniques: Immunohistochemical staining, Injection, Expressing, Luciferase, shRNA, Saline, Staining, Transgenic Assay, Immunofluorescence, Microscopy, Transduction, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Prothymosin α overexpression contributes to the development of pulmonary emphysema

doi: 10.1038/ncomms2906

Figure Lengend Snippet: ( a , b ) Immunohistochemical detection of MMP2 and MMP9 in lung tissues from patients with emphysema and non-COPD individuals (Scale bar=100 μm) ( a ) and from ProT HET and NT mice (Scale bar=20 μm) ( b ). ( c , e ) Expression of MMP2, MMP9, ProT and GAPDH (serving as the loading control) mRNA in lung tissues of 4-day-old ProT HZ, HET and NT mice ( c ) and in 293 T cells transduced with lentiviral vectors Lenti-ProT or Lenti-GFP ( e ), as analysed by RT–PCR. ( d ) Gelatin zymography of MMP2 and MMP9 in the conditioned medium of mouse embryonic fibroblasts (MEF) from ProT transgenic and NT mice. ( f , g ) Immunohistochemical detection of MMP9 in lung tissues (Scale bar=20 μm) from ProT HET and NT mice injected intraperitoneally with CSE or saline twice a week for 6 weeks ( f ) and from FVB mice that had been intratracheally injected with lentiviral vectors expressing ProT or luciferase (Luc) shRNA followed by intraperitoneal injections of CSE or saline twice a week for 6 weeks ( g ). The boxed areas in upper panels (× 400) are magnified and shown in lower panels ( a , b , f , g ). ( h ) ChIP analysis showing increases in NF-κB binding to MMP2 and MMP9 promoters by ProT overexpression. 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 were cotransfected with Flag-tagged NF-κB p65 expression vector or a control vector. Cross-linked chromatin was immunoprecipitated with anti-Flag-M2 beads and then amplified for the MMP2 or MMP9 gene region containing the NF-κB binding site. Cell lysates without immunoprecipitation (input) were estimated for chromatin containing MMP2 or MMP9 gene region. ( i ) ChIP analysis showing direct binding of ProT to the NF-κB-binding site-containing region of MMP2 and MMP9 promoters. 293T cells were transfected with plasmid Flag-tagged ProT (pCMV-Tag4A-ProT) or Flag control vector (left) or GFP-tagged ProT or GFP alone (right). Cross-linked chromatin was immunoprecipitated with anti-Flag-M2 beads (left) or anti-GFP or anti-IgG antibody combined with protein A plus G sepharose (right) followed by amplification of the MMP2 or MMP9 gene region containing NF-κB binding sites. Results are representative of three independent experiments.

Article Snippet: The primary antibodies used included monoclonal antibodies against ProT (clone 2F11, ascites fluid) and polyclonal antibodies against HDAC1 (1:100, Santa Cruz), HDAC2 (1:100, Santa Cruz), HDAC3 (1:100, Santa Cruz), acetylated-lysine (1:800, Cell Signaling), acetylated-Lys310 NF-κB p65 (1:100, Genetex), MMP2 (1:50, Santa Cruz) and MMP9 (1:50, Santa Cruz).

Techniques: Immunohistochemical staining, Expressing, Control, Transduction, Reverse Transcription Polymerase Chain Reaction, Zymography, Transgenic Assay, Injection, Saline, Luciferase, shRNA, Binding Assay, Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Amplification

Journal: Nature Communications

Article Title: Prothymosin α overexpression contributes to the development of pulmonary emphysema

doi: 10.1038/ncomms2906

Figure Lengend Snippet: ( a ) Healthy individuals who do not smoke cigarettes maintain the HAT/HDAC balance and thereby sustain normal transcription of NF-κB-dependent genes. ( b ) Non-smokers who have higher basal ProT levels exhibit reduced association of HDACs with histones in the lung. This leads to enhancing acetylation-mediated chromatin remodelling and thereby upregulating the expression of NF-κB-dependent genes, such as MMP2 and MMP9 . Thus, such individuals may develop mild emphysema. ( c ) In smokers who have normal ProT basal levels, exposure to CS induces ProT expression and nuclear translocation of NF-κB in the lung. This results in reduced HDAC activity and increased acetylation of chromatin and NF-κB, and in turn leads to upregulation of the transcription of NF-κB-dependent genes, such as MMP2 and MMP9 . This ultimately causes emphysema. Long-term cigarette smoking may exacerbate this effect and increase the severity of emphysema. ( d ) In smokers who have higher basal ProT levels, CS exposure further upregulates ProT expression. Excess ProT causes a shift in the HAT/HDAC balance toward HAT. This results in developing severe emphysema. Thus, ProT predisposes individuals to CS-induced emphysema.

Article Snippet: The primary antibodies used included monoclonal antibodies against ProT (clone 2F11, ascites fluid) and polyclonal antibodies against HDAC1 (1:100, Santa Cruz), HDAC2 (1:100, Santa Cruz), HDAC3 (1:100, Santa Cruz), acetylated-lysine (1:800, Cell Signaling), acetylated-Lys310 NF-κB p65 (1:100, Genetex), MMP2 (1:50, Santa Cruz) and MMP9 (1:50, Santa Cruz).

Techniques: Expressing, Translocation Assay, Activity Assay