man1a2 antibody  (Bio-Techne corporation)

Journal: PeerJ

Article Title: Circular RNA circMAN1A2 promotes ovarian cancer progression through the microRNA-135a-3p/IL1RAP/TAK1 pathway

doi: 10.7717/peerj.16967

Figure Lengend Snippet: The primer sequence of PCR and RT-qPCR.

Article Snippet: The primary antibody MAN1A2 (ab272611; Abcam) was diluted to the target ratio with antibody diluent and stored overnight at 4 °C.

Techniques: Sequencing

Journal: PLoS Pathogens

Article Title: N -Glycans and sulfated glycosaminoglycans contribute to the action of diverse Tc toxins on mammalian cells

doi: 10.1371/journal.ppat.1009244

Figure Lengend Snippet: (A) Five mammalian cell lines were treated with the indicated doses of PTC3 W14 for 24 h. Cell viability was measured using CCK-8 assays. The PTC3 W14 concentration that leads to the death of 50% of cells is defined as IC 50 . (B) HeLa-Cas9 cells transduced with a non-targeting sgRNA were treated with 5 nM PTC3 W14 for 0, 4 and 8 h. Cells were fixed, permeabilized and stained with Alexa 568-phalloidin (red) and DAPI (blue). Representative fluorescence and bright field micrographs are shown. White arrows indicate polymerized F-actin. Black arrows indicate membrane blebs. Scale bars, 5 μm. (C) Schematic drawing of the screening approach for host factors that are critical for the intoxication of PTC3 W14 . HeLa cells stably expressing Cas9 (HeLa-Cas9) were transduced with lentiviral GeCKO v.2 sgRNA libraries. These cells were exposed to increased doses of PTC3 W14 (5, 10 and 20 nM). Cells that were not treated with PTC3 W14 were served as controls. Total genomic DNA from 2×10 7 selected and control cells was used for sequencing. The enriched sgRNAs were sequenced by NGS, followed by MAGeCK analysis. (D) Genes identified in the screens with PTC3 W14 treatment were ranked based on to the MAGeCK p values. MAN1A2, MGAT1, and MGAT2 were identified as the key genes for PTC3 W14 recognition of HeLa cells. (E) The efficiency of gene knockout was determined by Western blot analysis with the indicated antibodies. GAPDH served as the loading control. (F-H) MAN1A2-KO, MGAT1-KO, and MGAT2-KO HeLa-Cas9 cells were treated with the indicated doses of PTC3 W14 for 24 h. Cell viability was measured using CCK-8 assays. (I-J) HeLa-Cas9-sgCon cells were pretreated with the indicated doses of Kifunensine for 12 h, and then exposed to 20 nM PTC3 W14 . Representative bright field micrographs were shown (I). Cell viability was measured using CCK-8 assays (J). All error bars indicate mean ± SD. Each of the experiments was repeated three times.

Article Snippet: Rabbit polyclonal anti-MAN1A2 antibody (PA5-56974, RRID: AB_2643718) was purchased from Thermo Fisher Scientific.

Techniques: CCK-8 Assay, Concentration Assay, Transduction, Staining, Fluorescence, Membrane, Stable Transfection, Expressing, Control, Sequencing, Gene Knockout, Western Blot

Journal: PLoS Pathogens

Article Title: N -Glycans and sulfated glycosaminoglycans contribute to the action of diverse Tc toxins on mammalian cells

doi: 10.1371/journal.ppat.1009244

Figure Lengend Snippet: (A-B) Representative bright field micrographs of indicated HeLa-Cas9 cells treated with indicated doses of PTC3 TT01 or PTC3 W14 (A). Cell viability was measured using CCK-8 assays and shown in (B). (C-D) MAN1A2-KO, MGAT1-KO, MGAT2-KO, C1GalT1-KO, and HeLa-Cas9-sgCon cells were exposed to 20 nM PTC3 TT01 . Representative bright field micrographs (C) and the effects on cell viability (D) were shown. (E) Schematic drawing of indicated TcAs. The N-terminal VRP1 domain (PF03538), the middle Neuraminidase domain (PF18413) and the C-terminal TcA_TcB_BD domains (PF18276) were shown. The RBD-D domains derived from TcdA1 TT01 and TcdA1 W14 are depicted in orange and green, respectively. (F-G) MAN1A2-KO, MGAT1-KO, MGAT2-KO, and HeLa-Cas9-sgCon cells were exposed to 20 nM of the indicated toxins. Representative bright field micrographs were shown (F). Cell viability was measured using CCK-8 assays (G). All error bars indicate mean ± SD. Each of the experiments was repeated three times.

Article Snippet: Rabbit polyclonal anti-MAN1A2 antibody (PA5-56974, RRID: AB_2643718) was purchased from Thermo Fisher Scientific.

Techniques: CCK-8 Assay, Derivative Assay

Journal: PLoS Pathogens

Article Title: N -Glycans and sulfated glycosaminoglycans contribute to the action of diverse Tc toxins on mammalian cells

doi: 10.1371/journal.ppat.1009244

Figure Lengend Snippet: (A) Schematic drawing of TcdA1 W14 , TcdA2 TT01 and TcdA1 W14 -RBD A2 . The RBD-D domains derived from TcdA1 W14 and TcdA2 TT01 are depicted in green and blue, respectively. (B-C) MAN1A2-KO, MGAT1-KO, MGAT2-KO, and HeLa-Cas9-sgCon cells were exposed to 20 nM of the indicated Tc toxins. Representative bright field micrographs (B) and the effects on cell viability (C) are shown. (D) Homology models of RBD-D domains. Swiss model program (ExPASy web server) was used to construct a homology model of the RBD-D of TcdA2 TT01 by alignment with that of TcdA1 W14 (PDB: 4O9Y). The Trp1721 and Lys1738 residues of TcdA1 W14 (corresponding to Ser1655 and Ser1672 of TcdA2 TT01 ) are indicated. (E-F) Representative bright field micrographs of indicated HeLa-Cas9 cells treated with the indicated doses of wild-type PTC3 W14 and indicated mutants (W1721S, K1738S, W1721S/K1738S) (E). Cell viability was measured using CCK-8 assays and shown in (F). All error bars indicate mean ± SD. Each of the experiments was repeated three times.

Article Snippet: Rabbit polyclonal anti-MAN1A2 antibody (PA5-56974, RRID: AB_2643718) was purchased from Thermo Fisher Scientific.

Techniques: Derivative Assay, Construct, CCK-8 Assay