Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. Western blot analysis of the expression levels of C16orf74 in pancreatic cancer cell lines. Control: Flag-tagged C16orf74-overexpressed diluted cell lysate. B. Phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by Western blot analysis using an anti-C16orf74 polyclonal antibody. The upper band disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). C. Phosphorylation at threonine 44 (T44) of C16orf74. Flag-tagged wild type (WT), T41A and T44A mutants of C16orf74 were used to transfect COS-7 cells. The phosphorylated form of wild-type C16orf74 (arrow) was disappeared in the T44A mutant. D. Immunocytochemical analysis in a pancreatic cancer cell line (PK-1) using the anti-C16orf74 antibody, demonstrating the plasma membrane localization of endogenous C16orf74 (Green). DAPI staining is shown in blue.

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: Western Blot, Expressing, Control, Incubation, Phospho-proteomics, Mutagenesis, Clinical Proteomics, Membrane, Staining

Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. In vitro exogenous association of C16orf74 and PPP3CA. The Flag-tagged C16orf74 construct or vector alone was cotransfected with a myc-tagged PPP3CA construct into HEK293 cells. Cell lysates were immunoprecipitated using mouse anti-Flag antibody (left) or anti-myc antibody (right). Immunoblotting of the immunoprecipitates with rabbit anti-Flag or anti-myc antibodies revealed a specific interaction between the phosphorylated form of C16orf74 (arrow) and PPP3CA. B. In vitro endogenous association of C16orf74 and PPP3CA from Capan-1 pancreatic cancer cells, which endogenously express high levels of both C16orf74 and PPP3CA. Capan-1 cell lysates were immunoprecipitated using anti-C16orf74 antibody (left) or anti- PPP3CA antibody (right). Immunoblotting of the immunoprecipitates with anti-C16orf74 antibody or anti-PPP3CA antibodies revealed a specific interaction between C16orf74 and PPP3CA. Endogenous PPP3CA interacted with the phosphorylated form of endogenous C16orf74 (arrow). C. Interactions of wild-type C16orf74 (WT) and mutants of C16orf74 with PPP3CA, as assessed by IP analysis. Expression vectors for myc-His-tagged PPP3CA and Flag-tagged C16orf74 constructs were doubly transfected into HEK293T cells. C16orf74 (anti-Flag) was IP, and the indicated molecules were immunoblotted (IB) in western blot analysis. WT, replacement (T44A; non-phosphorylated form of C16orf74) and deletion mutants (∆PDIIIT; deletion mutant of PPP3CA binding motif) were analyzed. PPP3CA bound to wild-type C16orf74 but not the non-phosphorylated form of C16orf74 or the deletion mutant of the PPP3CA binding motif. D. Subcellular localization of C16orf74 (wild type or ∆PDIIIT) and PPP3CA in mammalian cells. Flag-tagged (green) C16orf74 (wild type or ∆PDIIIT) and myc-tagged (red) PPP3CA constructs were cotransfected into COS-7 cells and subjected to immunocytochemical staining. Flag-C16orf74 (wild type) and myc-PPP3CA colocalized on the under the cytoplasmic membrane of COS-7 cells (yellow), but Flag-C16orf74 (∆PDIIIT) did not colocalize with myc-PPP3CA, which was present diffusely in the cytoplasm. E. Interactions of endogenous C16orf74 with PPP3CA as assessed by IP analysis. The phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by western blot analysis using an anti-C16orf74 polyclonal antibody. Pre IP (left; non-immunoprecipitated by PPP3CA), the phosphorylated form of C16orf74 (upper band) disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). Immunoprecipitation by PPP3CA (right) revealed that the phosphorylated form of C16orf74 (upper band) interacted with PPP3CA, whereas the non- phosphorylated form of C16orf74 did not. F. Invasion activity of wild-type C16orf74 (WT) and the two mutants (T44A: non-phosphorylated form of C16orf74; and ∆PDIIIT, deletion mutant of the PPP3CA binding motif). The WT-C16orf74 expression vector, T44A-C16orf74 expression vector, ∆PDIIIT-C16orf74 expression vector, and Mock vector were each transfected into NIH3T3 cells. The Matrigel invasion assay revealed an enhanced cell number for WT-C16orf74-over-expressing cells (3.4-fold, * P = 0.013) but not so enhanced for ∆PDIIIT-C16orf74-over-expressing cells (1.4-fold, ** P = 0.017) or T44A-C16orf74-over-expressing cells (2.3-fold,*** P = 0.038).

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: In Vitro, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Transfection, Mutagenesis, Binding Assay, Staining, Membrane, Incubation, Activity Assay, Invasion Assay

Journal: AIDS Research and Therapy

Article Title: The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients

doi: 10.1186/s12981-017-0158-7

Figure Lengend Snippet: Nucleotide sequences of the primers used for real-time PCR

Article Snippet: Antibodies (Abs) used in this study were as follows: mouse phycoerythin (PE)-conjugated anti-human IFN-alpha/beta R2 Ab (clone MMHAR-2, R&D Systems), mouse PE-conjugated anti-human IFN-lambdas R1 Ab (clone 601106, R&D Systems), mouse PE-conjugated anti-human IFN-gamma Receptor 1 (clone GIR-208, Thermo Fisher Scientific), Mouse eFluor ® 450-conjugated anti-human STAT1 (KIKSI0803, Thermo Fisher Scientific), Rabbit FITC anti-Human STAT2 (Thermo Fisher Scientific), Mouse PE-Cyanine7-conjugated anti-human STAT3 (clone LUVNKLA, Thermo Fisher Scientific), mouse APC-conjugated anti-human STAT4 (clone 4LURPLE, Thermo Fisher Scientific), mouse FITC-conjugated anti-human STAT5 (clone SRBCZX, Thermo Fisher Scientific), mouse PerCP-eFluor ® 710-conjugated anti-human STAT6 (clone CHI2S4N, Thermo Fisher Scientific).

Techniques: Sequencing

Journal: AIDS Research and Therapy

Article Title: The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients

doi: 10.1186/s12981-017-0158-7

Figure Lengend Snippet: Correlation between the CD4 + T cells and mRNA levels of IFN-alpha receptor ( a ), IFN-gamma receptor ( c ), and IFN-lambdas receptor ( e ). Correlation between the viral loads and mRNA levels of IFN-alpha receptor ( b ), IFN-gamma receptor ( d ), and IFN-lambdas receptor ( f ). The results were performed Spearman’s rank correlation, where coefficients “r” and corresponding p values are indicated on each panel

Article Snippet: Antibodies (Abs) used in this study were as follows: mouse phycoerythin (PE)-conjugated anti-human IFN-alpha/beta R2 Ab (clone MMHAR-2, R&D Systems), mouse PE-conjugated anti-human IFN-lambdas R1 Ab (clone 601106, R&D Systems), mouse PE-conjugated anti-human IFN-gamma Receptor 1 (clone GIR-208, Thermo Fisher Scientific), Mouse eFluor ® 450-conjugated anti-human STAT1 (KIKSI0803, Thermo Fisher Scientific), Rabbit FITC anti-Human STAT2 (Thermo Fisher Scientific), Mouse PE-Cyanine7-conjugated anti-human STAT3 (clone LUVNKLA, Thermo Fisher Scientific), mouse APC-conjugated anti-human STAT4 (clone 4LURPLE, Thermo Fisher Scientific), mouse FITC-conjugated anti-human STAT5 (clone SRBCZX, Thermo Fisher Scientific), mouse PerCP-eFluor ® 710-conjugated anti-human STAT6 (clone CHI2S4N, Thermo Fisher Scientific).

Techniques: