Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A-D) Human colonic EDMs were infected with Fn at moi 100 for 24 h. The RNA from EDMs was used for qRT-PCR to determine the expression of genes involved in base excision repair, mismatch repair and for non-homologous end joining (NHEJ). (A) Schematic showing the experimental design. (B) The level of BER transcripts, NEIL1, NEIL2, NTH1, OGG1, (C) The level of MMR transcripts, MLH1, MLH3, MSH2, MSH6, PMS2, (D) The transcript level of NHEJ marker Ku70 were determined by qRT-PCR. (E-F) Human colonic EDMs were infected with commensal E. coli -K12 strain (E), or pathogenic IBD-associated adherent invasive E. coli LF-82 (F) to determine the expression level of NEIL2 following infection. In (B-F), the expression level of the transcripts was normalized to the housekeeping gene (18srRNA), and the normalized expression value was compared with the respective uninfected control cells. Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, and ** indicates p≤0.01 as calculated by the unpaired two-tailed student’s t-test.

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Infection, Quantitative RT-PCR, Expressing, Non-Homologous End Joining, Marker, Control, Two Tailed Test

Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A) APC Min /+ EDMs derived from the uninvolved region of the colon were infected with different microbes; commensal E. coli K12, IBD-associated adherent-invasive E.coli LF82 and colon cancer-associated pathogens (NC101, H. pylori and Fn ). The supernatants were collected from the uninfected and infected EDMs done in the same experiments and assessed for oxidative DNA damage (right). Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, ** indicates p≤0.01 as assayed by student’s t-test. (B) The relative level of the oxidized bases produced by each microbe was compared with uninfected cells, which is considered as 1. The relative production of the oxidized base was compared between different microbes

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Derivative Assay, Infection, Produced

Journal: The Journal of Biological Chemistry

Article Title: Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase

doi: 10.1074/jbc.RA119.010999

Figure Lengend Snippet: Incorporation of nitroTyr into CaM expressed in E. coli. A, the human CaM construct with C-terminal His6 tag bearing either the native Tyr codon or an amber stop codon at amino acid position 99 or position 138 was co-transformed with a plasmid expressing an Methanocaldococcus jannaschii tyrosyl-tRNA synthetase/tRNACUA pair engineered to efficiently incorporate nitroTyr. B, WT– and nitroTyr–CaM was expressed in autoinduction medium with or without nitroTyr supplementation, purified using the C-terminal His6 tag, and analyzed by 15% SDS-PAGE gel. C, nitroTyr incorporation into CaM was determined here by Western blotting with a primary antibody against nitroTyr. D, electrospray ionization mass spectrometry confirms the quantitative incorporation of nitroTyr into CaM because all measured pure protein masses match their expected molecular masses.

Article Snippet: E. coli DH10B was transformed with pBad–CaM–(99 or 138 TAG) and pDule–nitroTyr–5B (Addgene plasmid no. 85498) ( 30 ).

Techniques: Construct, Transformation Assay, Plasmid Preparation, Expressing, Purification, SDS Page, Western Blot, Mass Spectrometry