ANIA:ANnotation and Integrated Analysis of the 14-3-3 interactome

The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate ‘lynchpins’, which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the ‘lynchpin’ site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology. Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.p

Western blotting guru /

Western Blotting Guru provides researchers in molecular biology with a handy reference for approaching and solving challenging problems associated with immunoblotting setup and optimization. As a laboratory guide, it emphasizes the technical aspects of efficiently employing immunoblotting as a tool in molecular biology laboratories. The book covers the basic science underlying immunoblotting and detailed description of the method parameters, followed by good benchtop practices, tips and tricks for obtaining high-quality data and a detailed troubleshooting guide addressing a variety of problem types.Includes bibliographical references.Front Cover; Western Blotting Guru; Copyright Page; Contents; Preface; Note to the Reader; 1 Introduction; 1.1 What Is Western Blotting?; 1.2 A Bit of History; 2 Procedure; 2.1 Sample Preparation; 2.2 Gel Electrophoresis; 2.2.1 Gel Selection and Preparation; 2.2.2 Running the Gels; 2.2.3 Molecular Weight Markers; 2.3 Protein Transfer; 2.3.1 Transfer Setup; 2.3.2 Choosing the Right Membrane; 2.3.3 Blotting Paper; 2.3.4 Transfer Buffer; 2.3.5 Transfer Power Settings; 2.3.6 Visualization of Proteins After Transfer; 2.4 Blocking the Membrane; 2.5 Primary Antibodies; 2.6 Secondary Antibodies.2.7 Blot Washes2.8 Developing Western Blots; 3 Good Practices; 4 Optimization and Troubleshooting; 4.1 Optimization Rules; 4.2 General Optimization Strategies; 4.2.1 Antibody Concentration; 4.2.2 Antibody Incubation Times; 4.2.3 Wash Stringency; 4.2.4 Blocking; 4.2.5 Preclear the Antibodies; 4.2.6 Supplement Antibody Solutions With Nonphosphorylated Peptides; 4.3 Troubleshooting Specific Problems; 4.3.1 Problem Type 1; 4.3.1.1 Subtype 1; 4.3.1.2 Subtype 2; 4.3.2 Problem Type 2; 4.3.3 Problem Type 3; 4.3.4 Problem Type 4; 4.3.5 Problem Type 5; 4.3.6 Problem Type 6; 4.3.7 Problem Type 7.4.3.8 Problem Type 84.3.9 Problem Type 9; 4.3.10 Problem Type 10; 5 Tips and Tricks; 6 Special Cases; 6.1 Quantitative Western Blotting; 6.2 Overlay Assays; 6.3 Phospho-Specific Antibodies; 6.4 Phos-Tag; 6.5 Nonreducing PAGE; 6.6 Dot Blots; 7 Data Analysis, Storage, Retrieval; Appendix A: Buffers and Solutions; Appendix B: SDS-PAGE Gel Tables; Appendix C: SDS-PAGE Protocol; Appendix D: Wet Transfer and Immunoblotting Protocol; Appendix E: Home-Made Enhanced ChemiLuminescence (ECL) Detection; Appendix F: Stripping Protocols; Appendix G: Coomassie Staining Protocol.Appendix H: Lysis of Cells Using Native ConditionsAppendix I: Quick Denaturing Lysis Protocol; Appendix J: Protein Tags; Appendix K: Covalent Crosslinking of Antibodies to Beads; References; Back Cover.Western Blotting Guru provides researchers in molecular biology with a handy reference for approaching and solving challenging problems associated with immunoblotting setup and optimization. As a laboratory guide, it emphasizes the technical aspects of efficiently employing immunoblotting as a tool in molecular biology laboratories. The book covers the basic science underlying immunoblotting and detailed description of the method parameters, followed by good benchtop practices, tips and tricks for obtaining high-quality data and a detailed troubleshooting guide addressing a variety of problem types.Elsevie

The effects of IRS4 overexpression on the PI3K pathway.

<p>(A) U2OS-Flpin™ cells expressing empty vector or IRS4-FLAG protein were analysed for the activation of PI3K pathway components under serum starved (SS), amino acid-starved (-AA) and 10% FBS conditions. Cells were deprived of serum for 12 h (SS) and starved of amino in EBSS for 2 h (-AA). (B) U2OS-Flpin™ cells expressing empty vector, full length IRS4-FLAG (WT), and the indicated fragments containing residues from 200–end-IRS4-FLAG or 336–end-IRS4-FLAG proteins were analysed for the activation of PI3K pathway components, in the presence or absence of serum. (C) As in B, except that the cells were treated with IGF1 (50 ng/ml, 20 min). The FLAG tagged proteins expressed were analysed for the Tyrosine phosphorylation as well as the binding to the p85 regulatory subunit of PI3 kinase.</p

Deletion of PH domain S4 targets IRS4 to the nucleus.

<p>U2OS-Flpin™ cells expressing full length IRS4-FLAG and the indicated fragments containing residues from 200–end-IRS4-FLAG or 336–end-IRS4-FLAG proteins were analysed for their subcellular localization, under serum starved (SS) or addition of IGF1(50 ng/ml, 20 min). Cells were stained for FLAG antibody and with DAPI as described in Materials and Methods.</p

HEK293 clones with increased IRS4 expression have elevated PI3K signalling.

<p>(A) Clones of HEK293 cells expressing different levels of IRS4 (#1-low, #2- and #3-high) obtained after retroviral infection and puromycin selection as described in materials and methods, were serum starved and then stimulated with IGF1 or 10% FBS. Cell lysates (30 µg) were subject to immunoblotting with the indicated antibodies. (B) As in (A), except that cells were starved of amino acids and then stimulated with amino acids or 10% FBS for 20 min. (C and D) The cells expressing different levels of IRS4 were serum deprived overnight and assayed for the catalytic activity of immunoprecipitated endogenous Akt and S6K1 using Crosstide peptide as substrate, as described in materials and methods. (E) Clones #1 and #3 of HEK293 cells were serum starved overnight and assayed for PIP₃ as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073327#pone.0073327-Gray1" target="_blank">[37]</a>. Cells from clone #1 were serum starved and treated with IGF1+/− PI-103 (1 µM, 30 min) and used as positive and negative controls, respectively. (F) Cells expressing low (#1) or high (#2 and #3) levels of IRS4 were transfected with control, IRS4 or IRS1 siRNA oligos and the activity of PI3K pathway components was analysed as in (A) under serum starved conditions.</p

Effects of IRS1 and IRS4 knockdown on the PI3K pathway in cells with low and high IRS4 protein levels.

<p>(A) Cell lines exhibiting high levels of IRS4 such as DMS114, NCI-H720, HEK293AAV, HEK293T were transfected with 100 nM final concentration of control (Scrambled), IRS4 or IRS1 siRNAs using Dharmafect 1 transfection reagent following manufacturer's instructions. After 48 h, cells were harvested and lysates (20 µg) were immunoblotted using the indicated antibodies to test the activation status of the PI3K pathway. (B) As in (A) except that the experiment was performed in cells with low expression of IRS4 such as HeLa, A431, HEK293, MDA-MB-231 and A549.</p

Gene synteny analyses of human IRS1, IRS2, IRS4 and the IRS3P pseudogene.

<p>A circle plot links the human gene synteny clusters containing human IRS1, IRS2, IRS4 genes and the IRS3P pseudogene. Plots were generated using the Synteny Database (teleost.cs.uoregon.edu/synteny_db) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073327#pone.0073327-Catchen1" target="_blank">[39]</a> with the <i>Branchiostoma</i> genome as outgroup. Arcs around the circumference represent chromosomes, while those within the circle connect pairs of related genes. The human IRS1 (chromosome 2q36), IRS2 (13q34) and IRS4 (Xq22.3) genes and also genes within 500 kb of these three genes, were used as queries. Different chromosomes were tested in the fourth position until a strong synteny trace was observed for chromosome 7 as shown, and these synteny lines were then confirmed to converge on the human IRS3P pseudogene at 7q22.1.</p