Journal: Cell chemical biology

Article Title: Split-miniSOG for spatially detecting intracellular protein-protein interactions by correlated light and electron microscopy

doi: 10.1016/j.chembiol.2019.07.007

Figure Lengend Snippet: (a) Wire diagram on the LOV2 domain (PDB: 4EEP) with relative B-factor values represented as a heat map (blue to red: 20 – 60 Å2) and also by wire diameter. B-factor values correspond to the displacement of atomic positions from the mean per atom, as calculated from crystallographic x-ray diffraction data. High B-values are associated with regions of high flexibility. FMN is shown as sticks. Arrows indicate the N-terminus, C-terminus, and the identified split-site (between Gly94 and Glu95). The image was generated using Chimera (https://www.cgl.ucsf.edu/chimera/). (b) Structure of the LOV2 domain (PDB: 2V0U) with residues corresponding to mSOG1–94 shown in purple, and residues corresponding to mSOG-Jα95–140 shown in green. FMN (spheres) makes direct contact with regions of both fragments. (c) While mSOG1–94 forms the bulk of the FMN binding-pocket (gray surface), residues on mSOG95–106/ mSOG-Jα95–140 (green) make contact with the cofactor through van der Waals interactions (residues 99 – 101, spheres) and through hydrogen bonding between the side-chain N of Gln103 (shown in sticks) and O4 of the FMN isoalloxazine ring. mSOG95–106 is a substructure of mSOG-Jα95–140 and lacks residues corresponding to the Jα-helix.

Article Snippet: Cat# 4084 Paraformaldehyde Electron Microscopy Science Cat# 19202 Glutaraldehyde Ted Pella Cat# 18426 Sodium cacodylate Ted Pella Cat# 18851 Glycine Bio-Rad Laboratories Cat# 161-0718 Diaminobenzidine (DAB, free base) Sigma-Aldrich Cat# D8001-10G Osmium tetroxide Electron Microscopy Science Cat# 19190 Durcupan ACM resin component A Sigma-Aldrich Cat# 44611 Durcupan ACM resin component B Sigma-Aldrich Cat# 44612 Durcupan ACM resin component C Sigma-Aldrich Cat# 44613 Durcupan ACM resin component D Sigma-Aldrich Cat# 44614 Critical Commercial Assays B-PER Bacterial Protein Extraction Reagent Pierce Cat#78248 Restore Western Blot Stripping Buffer Pierce Cat# 21059 Deposited Data EM data This paper Cell Image Library http://cellimagelibrary.org/project/P20094 Structure of A. sativa LOV2 domain Halavaty et al., 2007 PDB: 2V0U Structure of A. thaliana LOV2 domain Christie et al., 2012 PDB: 4EEP Experimental Models: Cell Lines HEK293 ATCC CRL-1573 HeLa ATCC CRM-CCL-2 HEK293T ATCC CVCL-0063 Experimental Models: Organisms/Strains Oligonucleotides Recombinant DNA pBAD/Myc-His A Invitrogen Cat# V440-01 pBAD-18cm Beckwith lab available through ATCC Cat# 87396 pQE-80L Qiagen Cat# 32943 pcDNA3.1 Invitrogen Cat# V790-20 pCAGGS (derived via restriction digest of the pCAGGSbased plasmid “PSD95-TS:YFP”) Tsien laboratory; PSD95-TS:YFP is available through AddGene Plasmid# 42225 Software and Algorithms Fiji Schindelin et al., 2012 https://imagej.net/Fiji Open in a separate window KEY RESOURCES TABLE Split-miniSOG allows to visualize intracellular protein-protein interactions Split-miniSOG complementation is reversible Reconstituted complexes are imaged by fluorescence microscopy and TEM/multicolor EM

Techniques: Generated, Binding Assay

Journal: Cell chemical biology

Article Title: Split-miniSOG for spatially detecting intracellular protein-protein interactions by correlated light and electron microscopy

doi: 10.1016/j.chembiol.2019.07.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cat# 4084 Paraformaldehyde Electron Microscopy Science Cat# 19202 Glutaraldehyde Ted Pella Cat# 18426 Sodium cacodylate Ted Pella Cat# 18851 Glycine Bio-Rad Laboratories Cat# 161-0718 Diaminobenzidine (DAB, free base) Sigma-Aldrich Cat# D8001-10G Osmium tetroxide Electron Microscopy Science Cat# 19190 Durcupan ACM resin component A Sigma-Aldrich Cat# 44611 Durcupan ACM resin component B Sigma-Aldrich Cat# 44612 Durcupan ACM resin component C Sigma-Aldrich Cat# 44613 Durcupan ACM resin component D Sigma-Aldrich Cat# 44614 Critical Commercial Assays B-PER Bacterial Protein Extraction Reagent Pierce Cat#78248 Restore Western Blot Stripping Buffer Pierce Cat# 21059 Deposited Data EM data This paper Cell Image Library http://cellimagelibrary.org/project/P20094 Structure of A. sativa LOV2 domain Halavaty et al., 2007 PDB: 2V0U Structure of A. thaliana LOV2 domain Christie et al., 2012 PDB: 4EEP Experimental Models: Cell Lines HEK293 ATCC CRL-1573 HeLa ATCC CRM-CCL-2 HEK293T ATCC CVCL-0063 Experimental Models: Organisms/Strains Oligonucleotides Recombinant DNA pBAD/Myc-His A Invitrogen Cat# V440-01 pBAD-18cm Beckwith lab available through ATCC Cat# 87396 pQE-80L Qiagen Cat# 32943 pcDNA3.1 Invitrogen Cat# V790-20 pCAGGS (derived via restriction digest of the pCAGGSbased plasmid “PSD95-TS:YFP”) Tsien laboratory; PSD95-TS:YFP is available through AddGene Plasmid# 42225 Software and Algorithms Fiji Schindelin et al., 2012 https://imagej.net/Fiji Open in a separate window KEY RESOURCES TABLE Split-miniSOG allows to visualize intracellular protein-protein interactions Split-miniSOG complementation is reversible Reconstituted complexes are imaged by fluorescence microscopy and TEM/multicolor EM

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Electron Microscopy, Protein Extraction, Western Blot, Stripping, Derivative Assay, Plasmid Preparation, Software

Journal: Annals of Translational Medicine

Article Title: Protective effect of hydrogen sulfide on endothelial cells through Sirt1-FoxO1-mediated autophagy

doi: 10.21037/atm-20-3647

Figure Lengend Snippet: Primary antibodies used

Article Snippet: Concentration used RRIDs Anti-GAPDH GAPDH fusion protein Ag0766 Proteintech, rabbit polyclonal antibody, 10494-1-AP 1:10,000 (WB) AB_2263076 Anti-LC3 Residues surrounding Leu44 of human LC3B protein (conserved in LC3A) Cell Signalling Technology, rabbit monoclonal antibody, #12741 1:1,000 (WB) AB_2617131 Anti-p62 P62/SQSTM1 fusion protein Ag13131 Proteintech, rabbit polyclonal antibody, 18420-1-AP 1:1,000 (WB) AB_10694431 Anti-ATG5 Residues surrounding Leu265 of human Atg5 protein Cell Signaling Technology, rabbit monoclonal antibody, #12994 1:1,000 (WB) AB_2630393 Anti-beclin-1 Beclin 1 fusion protein Ag1843 Proteintech, rabbit polyclonal antibody, 11306-1-AP 1:1,000 (WB) AB_2259061 Anti-Sirt1 A recombinant protein representing the central region of human SirT1 protein Cell Signaling Technology, mouse monoclonal antibody, #8469 1:1,000 (WB) AB_10999470 Anti-cleaved-caspase 3 Synthesized peptide derived from human caspase 3 Affinity Biosciences, rabbit polyclonal antibody, AF7022 1:1,000 (WB) AB_2835326 Anti-cleaved-PARP Synthetic peptide within human cleaved PARP1 aa 150–250 Abcam, rabbit monoclonal antibody, ab32064 1:1,000 (WB) AB_777102 FoxO1 Synthetic peptide mapping to a region between residues 600 and the C-terminus (residue 652) of human FOXO1A Abcam, rabbit polyclonal antibody, ab70382 1:500 (IF), 1:1,000 (WB) AB_1268912 Anti-acetylated-lysine A synthetic acetylated lysine-containing peptide Cell Signalling Technology, rabbit antibody, #9441 1:1,000 (WB) AB_331805 Anti-histone-H3 Histone-H3 fusion protein Ag10644 Proteintech, rabbit polyclonal antibody, 17168-1-AP 1:3,000 (WB) AB_2716755 Anti-tubulin Beta-tubulin fusion protein Ag0117 Proteintech, rabbit polyclonal antibody, 10068-1-AP 1:3,000 (WB) AB_2303998 Open in a separate window caption a8 Primary antibodies used

Techniques: Concentration Assay, Recombinant, Synthesized, Derivative Assay, Residue