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 cell lysates were sonicated with alternating 30 s pulses 10 times by a sonicator (Microson XL2000, Misonix Inc., Farmingdale, NY, USA) to shear DNA to lengths between 500 and 1,000 bp followed by immunoprecipitation with anti-Flag-M2 beads (Sigma-Aldrich), or with anti-GFP (Santa Cruz) or anti-IgG (Santa Cruz) antibodies in combination with protein A plus G sepharose (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