p53 prk5 vectors  (Addgene inc)

Journal: Frontiers in Immunology

Article Title: O- Linked N-Acetylglucosamine Modification of Mitochondrial Antiviral Signaling Protein Regulates Antiviral Signaling by Modulating Its Activity

doi: 10.3389/fimmu.2020.589259

Figure Lengend Snippet: MAVS contains a heavily O-GlcNAcylated serine-rich region that inhibits RLR signaling. (A) Fusion M/S analysis was conducted to identify sites of O-GlcNAcylation in MAVS. EThcD spectra of the O-glylcopeptide LPGPTGSVVSTGTSFSSSSPGLASAGAAEGK from human MAVS is as shown. The site of O-GlcNAc modification was identified as serine 249. The y, b, C, and z fragments detected are as indicated in the sequence. (B, C) MAVS O-GlcNAcylation sites and the protein structures of the wild-type and MAVS Δ249–257 mutant. (D, E) IP and Western blots were performed to compare the O-GlcNAcylation of wild-type MAVS with (D) the mutant in which the serine-rich region including the O-GlcNAcylation sites were detected and (E) the mutant with the 7S/T to alanine substitution. The pRK5-flag-tagged MAVS wild-type and mutant plasmids were transfected into HEK293 cells and evaluated 24 h later. WCLs were incubated with agarose-conjugated anti-FLAG antibody (M2) for 1–2 h at 4°C. (F, G) Western blot analysis of phospho-IRF3 (p-IRF3) in WCLs from HEK293 cells. The p-IRF3 levels were determined by transfection with pRK5-flag-tagged MAVS wild-type, (F) deletion mutant, or (G) substitution mutant plasmids followed by evaluation 24 h later; cells were infected with SeV (100 HAU) for 24 h followed by Western blotting. (H) Expression of IFN-β mRNA induced by overexpression of wild-type and mutant MAVS was measured by real-time qPCR. The decreased levels of IFN-β expression in response to the S366A mutant underwent full recovery in response to the Δ249–257 mutant containing S366A when compared to the wild type. n = 3. (D–H) Experiments were performed at least three times. (D–H) – indicated cells transfected with flag or myc empty vectors. Statistical significance of (H) was determined by one-way ANOVA. Results were presented as mean ± SEM; *p < 0.05, **p < 0.01.

Article Snippet: Wild-type and mutant forms of human MAVS wild type were prepared by PCR and cloned into the pRK5-Flag expression vector (Genentech).

Techniques: Modification, Sequencing, Mutagenesis, Western Blot, Transfection, Incubation, Infection, Expressing, Over Expression

Journal: Frontiers in Immunology

Article Title: O- Linked N-Acetylglucosamine Modification of Mitochondrial Antiviral Signaling Protein Regulates Antiviral Signaling by Modulating Its Activity

doi: 10.3389/fimmu.2020.589259

Figure Lengend Snippet: O-GlcNAcylation of MAVS interferes with aggregate formation and interactions with TRAF3. (A) Non-reducing SDD-PAGE analysis was conducted to determine the level of MAVS aggregation. MAVS overexpression in HEK293 cells induced aggregate formation levels with or without overexpression of OGA. (B) Knock-down of OGT in HEK293 cells was detected at 48 h after transfection with specific siRNAs; this was performed before induction of MAVS overexpression for 24 h, after which SDD-AGE analysis was performed. (C) Endogenous MAVS aggregates were detected by SDD-AGE under conditions of OGT knock-down. HEK293 cells were transfected with siOGT; 48 h later, cells were infected with SeV (100 HAU) and evaluated at 16 h after infection. MAVS aggregates were normalized to the expression level of endogenous MAVS. (D) Precipitation was performed with sWGA followed by Western blot analysis to detect the O-GlcNAcylation levels of MAVS after a longer period of time after SeV infection (100 HAU). Cells were infected with SeV for 24 h before collection. WCLs were incubated with sWGA-conjugated beads overnight at 4°C and then were eluted for Western blot analysis. (E) An SDD-AGE assay was conducted to compare the levels of aggregation of wild-type MAVS and mutants with deleted O-GlcNAcylation sites. The pRK5-flag-tagged MAVS wild-type and mutant (Δ249–257, S366A, Δ249–257 containing S366A) plasmids were used to transfect HEK293 cells and were evaluated after 24 to 48 h. (F) A co-IP assay was performed to evaluate the interactions between MAVS and TRAF3. The pRK5-flag-tagged MAVS and HA-TRAF3 expression plasmids co-overexpressed in HEK293 cells both with and without Myc-OGT overexpression. (G) A co-IP assay was performed to evaluate the interactions between wild-type or the Δ249–257 mutant MAVS and HA-TRAF3 in HEK293 cells. Expression plasmids were used to transfect cells that were evaluated at 24 to 48 h. All experiments were repeated at least three times. (A–G) – means cells transfected with flag or Myc or HA empty vectors.

Article Snippet: Wild-type and mutant forms of human MAVS wild type were prepared by PCR and cloned into the pRK5-Flag expression vector (Genentech).

Techniques: Over Expression, Transfection, Infection, Expressing, Western Blot, Incubation, Mutagenesis, Co-Immunoprecipitation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: To generate LUZP1 KO, ZO‐1/‐2 DKO, and E‐cadherin KO Eph4 cells, we used the CRISPR/Cas9 system with the pX330 vector (#42330; Addgene) to knockout mouse LUZP1, ZO‐1, and E‐cadherin genes.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification