Journal: The Journal of Cell Biology

Article Title: A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity

doi: 10.1083/jcb.201308032

Figure Lengend Snippet: Yrt is a substrate of aPKC. (A) Control embryos ( daughterless [ da ] -GAL4 ) or embryos overexpressing Par-6 and aPKC CAAX ( da-GAL4/UAS–Par-6 and UAS-aPKC CAAX ) were homogenized at different developmental stages. Samples were treated or not treated with the λ phosphatase (λ PPase) and processed for SDS-PAGE. Western blotting using anti-Yrt antibodies showed the migration profile of Yrt, whereas Actin was used as loading control. (B) Western blot showing the migration profile of Yrt extracted from control (wild type) or aPKC maternal and zygotic mutant embryos (we used the allele aPKC psu265 that encodes a kinase inactive protein; ). Actin was used as a loading control. (C and D) Radioactive kinase assays in which purified aPKC was incubated with GST coupled to full-length Yrt (FL; C) or an extended version of the FA domain of Yrt (FA; aa 330–415) and a mutant version of it in which S348, S358, T379, S387, and S392 were mutagenized to A residues (FA 5A ; D). Proteins were separated on a polyacrylamide gel, which was exposed to monitor protein phosphorylation and then colored with Coomassie blue to control the amount of substrate used in each sample. (E) Anti–Par-6 antibodies were used to immunoprecipitate Par-6 from wild-type embryo extracts (stages 10–13; immunoprecipitate [IP] Par-6), whereas normal guinea pig IgG (IgG) was used as a negative control. GST or GST-FA was added to precipitate along with radiolabeled ATP in the absence or presence of PKCtide, which is a high affinity substrate of aPKC. Proteins were separated by SDS-PAGE, and the gel was exposed to monitor protein phosphorylation. Then, proteins were transferred on a membrane to validate immunoprecipitation of Par-6 and coimmunoprecipitation of aPKC and to monitor the amount of substrate used in each reaction. (F) GST pull-down experiments were performed on wild-type embryo lysates using GST-FA (FA), the nonphosphorylatable GST-FA 5A (FA 5A ), or the phosphomimetic GST-FA 5D (FA 5D ). GST alone was used as a negative control. Western blotting detected pulled down aPKC and monitored the amount of GST or GST fusion proteins used in each experiment. (G) Alignment of the FA domain of mouse Lulu2 and Drosophila Yrt. Numbers indicate amino acid positions in the Yrt sequence. Arrows point to amino acids previously shown to be phosphorylated by aPKC in Lulu2 . Three of these residues are conserved in Yrt (black rectangles). Black circles indicate phosphorylated residues identified by MS in the FA domain of Yrt. Three of these residues are conserved in Lulu2 (orange rectangles). As per ClustalW nomenclature , asterisks indicate positions that have fully conserved residues. Colons designate conservation between groups of strongly similar properties, and periods indicate conservation between groups of weakly similar properties.

Article Snippet: For λ phosphatase assays, embryos were lysed in lysis buffer without phosphatase inhibitors. λ Phosphatase (New England Biolabs, Inc.) was used following the manufacturer’s recommendations.

Techniques: SDS Page, Western Blot, Migration, Mutagenesis, Purification, Incubation, Negative Control, Immunoprecipitation, Sequencing

Journal: Biosensors

Article Title: Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

doi: 10.3390/bios12080569

Figure Lengend Snippet: Feasibility of M.SssI detection with RCA. ( A ) Fluorescence emission spectra with or without M.SssI following the addition of SYBR TM Gold. ( B ) Polyacrylamide gel electrophoresis supports the feasibility of the strategy: (Lane M) 20 bp DNA marker; (Lane a) PPs; (Lane b) PPc; (Lane c) PPs + PPc + T4 DNA ligase; (Lane d) PPs + PPc + T4 DNA ligase + HpaII + lambda exo + ExoI; (Lane e) PPs + PPc + T4 DNA ligase + M.SssI + HpaII + lambda exo+ ExoI.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/bios12080569/s1 , Table S1: Oligonucleotides used in this study; Table S2: Detection limits and real sample applications of some MTase detection methods; Figure S1: Electrophoresis analysis of the products of RCA of the detection system with (Lane a) and without (Lane b) M.SssI by RCA (Lane M) 15,000 bp DNA marker; Figure S2: Optimize the methylation and cleavage buffers. (A) F is the fluorescence intensity with M.SssI and F 0 is fluorescence intensity without M.SssI. (B) The fluorescence intensities of the sensing systems with or without M.SssI. (a) CutSmart buffer (methylation process) + CutSmart buffer (cleavage process); (b) NEBuffer 2 (methylation process) + CutSmart buffer (cleavage process); (c) NEBuffer 2(methylation process) + CutSmart buffer + lambda reaction buffer (cleavage process); (d) CutSmart buffer (methylation process) + lambda reaction buffer (cleavage process); Figure S3: Optimize the time of methylation process; Figure S4: Optimize the time of cleavage of HpaII and lambda exo; Figure S5: Optimize the time of digestion of ExoI; Figure S6: Eliminate the probable impact of detection system.

Techniques: Fluorescence, Polyacrylamide Gel Electrophoresis, Marker