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: The Journal of Neuroscience

Article Title: Trip6 Promotes Dendritic Morphogenesis through Dephosphorylated GRIP1-Dependent Myosin VI and F-Actin Organization

doi: 10.1523/JNEUROSCI.2125-14.2015

Figure Lengend Snippet: GRIP1956T is phosphorylated by AKT1. A, GFP-LR2+PDZ7 (GRIP1 fragment) was immunoprecipitated (IP) with anti-GFP antibody and separated by SDS-PAGE followed by mass spectrometry analysis. Threonine at the 956 position (956T) was identified to be phosphorylated (red box). Co. St., Coomassie blue staining. B, Alignment of the amino acid sequence containing GRIP1855T (negative control) and GRIP1956T among different eukaryotes; 956T is emphasized in bold and marked by an asterisk; the predicted AKT1-phosphorylated motif is indicated by the arrow. C, Overexpressed GFP-LR2+PDZ7 (GRIP1 fragment) was immunoprecipitated from HEK293T cells with anti-GFP antibody and treated with lambda phosphatase (λ PPase) or calf intestine alkaline phosphatase (CIAP) followed by immunoblotting (IB) with anti-GRIP1956T phosphorylation-specific antibody (pGRIP1). D, WT and phosphodead mutants T855A, T956A, and both T855A and T956A (T855A+T956A) of GFP-LR2+PDZ7 were transfected into HEK293T cells and analyzed by IB with pGRIP1 antibody. E, F, IB of phospho-GRIP1956T (pGRIP1) and total GRIP1 (tGRIP1) in the whole-brain (E) and hippocampus (F) homogenates of mice at different developmental stages. E, Embryonic day; P, Postnatal day; PW, Postnatal week. G, Pull-down of overexpressed GFP-AKT1 by GST-LR2+PDZ7 in HEK293T cell lysates. H, GFP-LR2+PDZ, together with AKT1 shRNA vector (628 or 642), was transfected into HEK293T cells for 72 h followed by IB. I, J, HEK293T cells overexpressing WT (I) or T956A (J) of GFP-LR2+PDZ7 were treated with insulin alone or together with wortmannin following serum starvation for 36 h and then analyzed by IB with pGRIP1 antibody. K, Cultured hippocampal neurons were treated with insulin alone or together with wortmannin following serum starvation at 7 DIV for 36 h and then analyzed by IB with pGRIP1 antibody.

Article Snippet: One aliquot was left untreated, and the other two were washed extensively with their respective buffers before treatment either with 400 U lambda phosphatase (Merck) at 30°C for 30 min or with 400 U of calf intestinal alkaline phosphatase (Promega) at 37°C for 30 min.

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Staining, Sequencing, Negative Control, Western Blot, Transfection, shRNA, Plasmid Preparation, Cell Culture