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: bioRxiv

Article Title: Lysosomal MLKL is balanced by ESCRT to control cell death

doi: 10.1101/2023.08.29.555049

Figure Lengend Snippet: ( a ) Western blot analysis of SH-SY5Y cells primed for 16 h with IFN type I followed or not by treatment with Poly(I:C) (IFN/pIC) for 48 h using anti-MLKL, -RIPK3 and -actin antibodies. ( b and c ) MLKL was knocked out by CRISPR/Cas9 approach using two different gRNAs in SH-SY5Y cells. ( b ) Western blot analysis of MLKL expression levels using anti-MLKL and -actin antibodies treated with IFN for 16 h. ( c ) Cell death profile following IFN/pIC treatment for 48 h analyzing SytoxGreen (SG) positive cells. Data points represent the mean ± □S.E.M. of n = 3. **p = 0.009 and 0.0032. ( d ) Cell death profile analyzing PI positive SH-SY5Y cells, left untreated or treated for 48 h with IFN/pIC in combination with GSK-872’ (RIPK3i) or necrosulfonamide (NSA). Data points represent the mean ± S.E.M. of four independent experiments (n = 4); ***p=0.0004; ns= not significant. ( e ) Phostag SDS-Page of SH-SY5Y cells treated for indicated times with IFN/pIC or HT-29 cells with pBZ for 12 h to induce necroptosis using anti-p-MLKL (T357/S358) or anti-MLKL antibody. Phospho-MLKL bands were verified by lambda phosphatase treatment. A representative of n = 3 is shown. ( f ) Western blot analysis under non-reducing (-DTT) and reducing (+DTT) conditions of SH-SY5Y cells treated with IFN/pIC for the indicated times using an anti-MLKL antibody. A representative of n=3 is shown; left panel. Oligomeric MLKL is indicated. Right panel: Western blot analysis under non-reducing conditions of HT-29 cells treated with pBZ (poly(I:C), BV6 and zVAD-fmk; necroptosis (nec)) for 12 h using an anti-MLKL antibody. Oligomeric MLKL is indicated by an arrowhead. b: bottom of the well; i: interface between stacking and running gel. ( g ) Confocal images of MLKL-GFP expressing SH-SY5Y cells expressing LAMP-1-RFP and Hoechst left untreated or treated for 24 h with IFN/pIC. Merged image of MLKL-GFP, LAMP-1 and Hoechst right panel. Scale bars 10 µm. Representative images of n = 1 are shown. ( h ) Quantification of ( g ) analyzing the percentage of overlay of LAMP-1-RFP fluorescence intensity with MLKL-GFP. Data points represent the mean ± S.E.M. of n = 2 analyzing at least 25 cells. ****p < 0.0001.

Article Snippet: For lambda phosphatase treatment cells were lysed in RIPA buffer and incubated with lambda phosphatase (New England biolabs; P0753S) for 30 min at 37 °C prior to TCA precipitation and loading on Phostag SDS-Page.

Techniques: Western Blot, CRISPR, Expressing, SDS Page, Fluorescence

Journal: STAR Protocols

Article Title: Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

doi: 10.1016/j.xpro.2021.100588

Figure Lengend Snippet:

Article Snippet: T4 DNA Ligase Reaction Buffer , NEB , Cat# B0202S.

Techniques: Recombinant, Lambda DNA Preparation, Staining, Software, Confocal Microscopy, Protein Purification, Chromatography