Journal: British Journal of Cancer

Article Title: A protein fragment derived from DNA-topoisomerase I as a novel tumour-associated antigen for the detection of early stage carcinoma

doi: 10.1038/bjc.2016.369

Figure Lengend Snippet: Detection of expression and cellular location of the novel TAA by fluorescence immunostaining and immunohistochemistry. ( A ) The expression and cellular location of the novel TAA in the MBA-MD-231 breast cancer cell line and acute leukaemia were examined by using fluorescence immunostaining with rabbit polyclonal antibody against the novel TAA. Original magnification at × 100. ( B ) The expression and cellar location of the novel TAA were detected by immunohistochemistry in various types of human carcinoma. Original magnification at × 200.

Article Snippet: To obtain the immunoblot with the rabbit polyclonal antibody against the novel TAA or the anti-SCL-70 autoantibodies (Meridian Life Science), the recombinant human TOP1 fragments were separated on the SDS–PAGE and electroblotted to the PVDF membranes as described above.

Techniques: Expressing, Fluorescence, Immunostaining, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: A protein fragment derived from DNA-topoisomerase I as a novel tumour-associated antigen for the detection of early stage carcinoma

doi: 10.1038/bjc.2016.369

Figure Lengend Snippet: Comparison of immunoreactivity of the novel TAA with several autoantibodies. ( A ) Comparison of the immune activity against the purified novel TAA in serum samples from healthy controls (Con), cancer patients and patients with positive autoantibodies against CK19, cyclic citrullinated peptide (CCP), dermatomyositis (DM), islet cells, double-stranded DNA (dDNA), histamine, phosphatidylethanolamine (PE), ribosome, p53 and extractable nucleus (EN). The results represent the mean+s.e. for each group (* P <0.001 as compared with controls). ( B ) Comparison of the immune activity of the autoantibodies against the novel TAA with the autoantibodies against RNP, SS-A, SS-B, Jo1, SM and SCL-70. The results represent the mean+s.e. for each group (* P <0.001 as compared with healthy controls). ( C ) Comparison of the autoantibodies against the novel TAA with the anti-SCL-70 autoantibodies and the rabbit polyclonal antibody. The novel TAA (2 μ g ml -1 , 50 μ l per well) was coated onto microtiter plates. 1 : 1000 dilution of the rabbit polyclonal antibody was incubated together with various concentrations of the anti-SCL-70 autoantibodies. The results represent the mean+s.e. for each group (* P <0.001 as compared with controls without the anti-SCL-70 autoantibodies).

Article Snippet: To obtain the immunoblot with the rabbit polyclonal antibody against the novel TAA or the anti-SCL-70 autoantibodies (Meridian Life Science), the recombinant human TOP1 fragments were separated on the SDS–PAGE and electroblotted to the PVDF membranes as described above.

Techniques: Comparison, Activity Assay, Purification, Incubation

Journal: British Journal of Cancer

Article Title: A protein fragment derived from DNA-topoisomerase I as a novel tumour-associated antigen for the detection of early stage carcinoma

doi: 10.1038/bjc.2016.369

Figure Lengend Snippet: Identification of the novel TAA as a fragment of human DNA-topoisomerase I. ( A ) Western blot analysis of the anti-SCL-70 autoantibody reactions with four recombinant fragments of TOP1. ( B ) ELISA analysis of the anti-SCL-70 autoantibody reactions with four recombinant fragments of TOP1. The results represent the mean+s.e. for each group (* P <0.001). ( C ) Western blot analysis of the rabbit polyclonal antibody reactions with four recombinant fragments of TOP1. ( D ) ELISA analysis of the rabbit polyclonal antibody reactions with four recombinant fragments of TOP1. The results represent the mean+s.e. for each group (* P <0.001). ( E ) Comparison of serum immune reaction to recombinant TOP1 fragments among cancer patients, scleroderma patients and healthy donors. The immune reactions in the serums of the cancer patients and scleroderma patients are presented as percentages of mean+s.e. relative to the serums of the healthy controls for each fragment. The P -values indicate the comparisons of the immune reactions in serums between the cancer patients and the scleroderma patients to the each recombinant fragment. ( F ) One hundred and eighty serum (180) samples were collected from cancer patients, healthy donors and other non-cancer patients. Serum autoantibody against the recombinant TOP1 amino acid sequence 329–765 fragment and the chromatographically purified novel TAA were determined simultaneously, with the correlation being analysed using a linear regression technique. A strong correlation was observed ( y =0.00062+0.844 X , r =0.929, P< 0.0001). ( G ) Comparison of antibody affinity of the rabbit anti-novel TAA antibody to the recombinant TOP1 amino acid sequence 329–765 fragment and the chromatographically purified novel TAA. The results represent the mean+s.e. for each group (* P <0.001).

Article Snippet: To obtain the immunoblot with the rabbit polyclonal antibody against the novel TAA or the anti-SCL-70 autoantibodies (Meridian Life Science), the recombinant human TOP1 fragments were separated on the SDS–PAGE and electroblotted to the PVDF membranes as described above.

Techniques: Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Comparison, Sequencing, Purification

Journal: British Journal of Cancer

Article Title: A protein fragment derived from DNA-topoisomerase I as a novel tumour-associated antigen for the detection of early stage carcinoma

doi: 10.1038/bjc.2016.369

Figure Lengend Snippet: Association of the autoantibodies against the novel TAA with early-stage carcinomas. ( A ) Serum levels of the autoantibodies against TOPO48 in healthy controls and patients with early and late stages of ESSC, GC, CRC and NSCLC were assessed by using the ELISA. Median levels of serum anti-TOPO48 autoantibody ranges are illustrated by box plot. The maximum value of the immune activity in the healthy controls was defined as a cutoff value (horizontal short dash line). The P -values indicate the comparisons of the immune reactions in serums between the early and late stages of each type of carcinoma. ( B ) Percentages of serum samples with positive TOPO48 autoantibody in early stages (I/II) and late stages (III/IV) of ESCC, GC, CRC and NSCLC. The cutoff point was defined as 1.42 U ml -1 . The P -values indicate the comparisons of the percentage of serum samples with positive TOPO48 autoantibody between the early and late stages of each type of carcinoma. ( C–F ) ROC analysis for the autoantibody levels for healthy controls versus early stage NSCLC, GC, CRC and ESCC, respectively.

Article Snippet: To obtain the immunoblot with the rabbit polyclonal antibody against the novel TAA or the anti-SCL-70 autoantibodies (Meridian Life Science), the recombinant human TOP1 fragments were separated on the SDS–PAGE and electroblotted to the PVDF membranes as described above.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: (A) Structural model DOCK2 generated by docking simulations followed by MD refinement carried out at full atomic scale in explicit membrane (gray sticks) and water. The simulations were performed for the intact γ-secretase complex that displays tight interaction between APH1A (magenta) and βarr2 (salmon) and transient involvement of other γ-secretase complex subunits such as the PS1 (cyan) and NCT (green). (B) Detailed view of interfacial interactions robustly observed at the interface. Residues engaged in persistent interfacial contacts are shown as sticks and labeled. (C) Interactions between APH1A and βarr2 for two independent runs (40 ns each) were carried out for this model (the first two columns) and corresponding time evolution of interactions for both runs. The last column displays the cumulative fractional time during which those pairs made contacts. Here, interactions are defined when any pairs of heavy atoms belonging to the two respective proteins are separated by less than 5 Å. Equivalent results for two other models, termed Alignment and DOCK1, are presented in , supporting the robustness of the regions (ICL2 and ICL3 in APH1A and finger loop and C terminus in βarr2) and residues engaged in interfacial association, despite minor redistributions of the specific pairs of residues.

Article Snippet: Rabbit polyclonal antibodies to human PS1-NTF (B19.3, 1:20,000), APH1A L (B82.3, 1:1,000), PEN-2 (B126.2, 1:1,000) and the APP C terminus (B63.3, 1:10,000) were the gift of Dr. Bart De Strooper (VIB and KU Leuven, Leuven, Belgium; UK Dementia Research Institute and University College London, London, United Kingdom) ( ; ).

Techniques: Generated, Membrane, Labeling

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal antibodies to human PS1-NTF (B19.3, 1:20,000), APH1A L (B82.3, 1:1,000), PEN-2 (B126.2, 1:1,000) and the APP C terminus (B63.3, 1:10,000) were the gift of Dr. Bart De Strooper (VIB and KU Leuven, Leuven, Belgium; UK Dementia Research Institute and University College London, London, United Kingdom) ( ; ).

Techniques: Recombinant, PathHunter β-Arrestin Assay, Mutagenesis, BIA-KA, Software, Membrane