Journal: British Journal of Cancer

Article Title: Identification of GlcNAcylated alpha-1-antichymotrypsin as an early biomarker in human non-small-cell lung cancer by quantitative proteomic analysis with two lectins

doi: 10.1038/bjc.2015.348

Figure Lengend Snippet: Quantitative LC-MS/MS and N-linked glycosylation analysis of AACT, AGP1, CFB, HPX proteins. ( A – D ) Left , the amino-acid sequences ADLSGITGAR, NWGLSVYADKPETTK, AVISPGFDVFAK and DYFWPCPGR were used to identify AACT, AGP1, CFB and HPX, respectively; Right , the released iTRAQ reporter ions were used to provide the relative quantitation of each protein (Healthy, 114 isobaric tag; Benign, 115 isobaric tag; Stage IA, 116 isobaric tag; Stage IB, 117 isobaric tag). ( E – H ) Five pooled healthy serum samples were treated with or without PNGase F and analysed by immunoblotting for anti-AACT, anti-AGP1, anti-CFB and anti-HPX antibodies. −, PNGase F-untreated crude samples; +, PNGase F-treated crude samples. A full colour version of this figure is available at the British Journal of Cancer journal online.

Article Snippet: For western blots, the membranes were blocked in 3% BSA in 0.1% TBST and probed with mouse monoclonal antibody against AACT (1 : 5000; Protein-tech, Wuhan, China), and rabbit polyclonal antibodies against AGP1 (1 : 5000; Protein-tech), CFB (1 : 5000; Protein-tech) and HPX (1 : 5000; Protein-tech) as primary antibodies, overnight at 4 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Western Blot

Journal: British Journal of Cancer

Article Title: Identification of GlcNAcylated alpha-1-antichymotrypsin as an early biomarker in human non-small-cell lung cancer by quantitative proteomic analysis with two lectins

doi: 10.1038/bjc.2015.348

Figure Lengend Snippet: Verification of selected protein candidate biomarkers by western blotting, ROC curve analysis, immunohistochemistry and animal model studies. ( A – D ) Densitometry analysis of western blots of 66 individual sera. The densitometric ratios were calculated as the value for an individual sample divided by the normalisation control value (positive control samples: H1). ( A ) AACT, ( B ) AGP1, ( C ) CFB, ( D ) HPX. * P <0.05; ** P <0.01; *** P <0.001. ( E, F ) ROC curves for differentiating Stage IA (E) or Stage (IA+IB) (F) from HB samples. ( G ) The performance of candidate proteins in distinguishing early NSCLC are listed. Grey bold indicates the improved performance of individual protein or combined proteins. ( H ) Immunohistochemistry analysis of AACT and AGP1 in Stage IB AD patients compared with normal lung tissue specimens. Upper , AACT (no haematoxylin staining), original magnification, × 40; under , AGP1 (haematoxylin staining), original magnification, × 200. (I) Serum AGP1 protein levels were reduced in the sera of LLC mice ( n =14).

Article Snippet: For western blots, the membranes were blocked in 3% BSA in 0.1% TBST and probed with mouse monoclonal antibody against AACT (1 : 5000; Protein-tech, Wuhan, China), and rabbit polyclonal antibodies against AGP1 (1 : 5000; Protein-tech), CFB (1 : 5000; Protein-tech) and HPX (1 : 5000; Protein-tech) as primary antibodies, overnight at 4 °C.

Techniques: Western Blot, Immunohistochemistry, Animal Model, Positive Control, Staining