Journal: The FASEB Journal

Article Title: Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection

doi: 10.1096/fj.201901290R

Figure Lengend Snippet: SDS-PAGE and Western blotting of rATP5A1 and its binding to gram-positive and gram-negative bacteria, LTA, LPS, and lipid A. A) SDS-PAGE. Lane M, marker; lane 1, negative control (empty vector pET28a); lane 2 and 3, total cellular extracts from E. coli BL21 containing expression vector pET28a/atp5a1 before induction (2) or induced with IPTG (3); lane 4, purified rATP5A1; 5, Western blotting of rATP5A1. B, C) Western blotting about interaction of rATP5A1 with gram-positive (B) and gram-negative (C) bacteria. Lane M, marker; lane 1, purified rATP5A1; lane 2, 4, and 6, M. luteus, B. subtilis, and S. aureus (B) or E. coli, V. anguillarum, and A. hydrophila (C) incubated in the presence of rATP5A1; lane 3, 5, and 7, M. luteus, B. subtilis, and S. aureus (B) or E. coli, V. anguillarum, and A. hydrophila (C) incubated in the absence of rATP5A1. D) No affinity of TRX-His-tag peptide with the gram-positive and gram-negative bacteria. Lane M, marker; lane 1, purified TRX-His-tag; lane 2, 3, 4, 5, 6, and 7, M. luteus, B. subtilis, S. aureus, E. coli, V. anguillarum, and A. hydrophila incubated in the presence of TRX-His-tag peptide. E, F). Binding of FITC-labeled rATP5A1 to gram-positive (Ea, d, g) and gram-negative (Fa, d, g) bacteria; binding of FITC-labeled rATP5A1 preincubated with LTA to gram-positive bacteria (Eb, e, h) or preincubated with LPS to gram-negative bacteria (Fb, e, h); no binding of FITC-labeled TRX-His-tag peptide to gram-positive (Ec, f, i) and gram-negative (Fc, f, i) bacteria. Scale bars, 20 μm. G, H, I) Interaction of rATP5A1 with LTA (G), LPS (H), and lipid A (I) revealed by ELISA. J) Effects of various sugars on the interaction of rATP5A1 with LPS. Each point in the graph represents the mean ± sd (n = 3). The data are from 3 independent experiments performed in triplicate. The bars represent means ± sd. GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; OD, optical density.

Article Snippet: The PCR product was digested with Eco RI (Takara) and Hind III (Takara) and subcloned into the pET28a expression vector (Merck GmbH, Darmstadt, Germany) digested previously with the same restriction enzymes.

Techniques: SDS Page, Western Blot, Binding Assay, Marker, Negative Control, Plasmid Preparation, Expressing, Purification, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection

doi: 10.1096/fj.201901290R

Figure Lengend Snippet: Diagram showing zebrafish ATP5A1 truncation, SDS-PAGE, and Western blotting of rA1–65, rA66–134, and rA135–551 and their antibacterial activities and binding to LPS and LTA. A) Diagram showing zebrafish ATP5A1 truncation. B) SDS-PAGE and Western blotting of rA1–65 (a), rA66–134 (b), and rA135–551 (c). Lane M, marker; lane 1, negative control (empty vector pET28a); lane 2, total cellular extracts from E. coli BL21 containing expression vector pET28a/a1-65 (a), pET28a/a66-134 (b), and pET28a/a135-551 (c) before induction; lane 3, total cellular extracts from IPTG-induced E. coli BL21 containing expression vector pET28a/a1-65 (a), pET28a/a66-134 (b), and pET28a/a135-551 (c); lane 4, purified rA1–65 (a), rA66–134 (b), and rA135–551 (c); lane 5, Western blotting of rA1–65 (a), rA66–134 (b), and rA135–551 (c). COOH, free carboxyl end group; H2N, free amino end group. C) Antibacterial activity of rA1–65 against M. luteus, B. subtilis, S. aureus, E. coli, V. anguillarum, and A. hydrophila. D–F) Interaction of rA1–65 (D), rA66–134 (E), and rA135–551 (F) with LTA and LPS revealed by ELISA. G) The in vivo bioactivity of rA1–65, rA66–134 and rA135–551. The early (8-cell stage) embryos were first microinjected with PBS, BSA, AcAb, ATP5A1Ab, rA1–65, rA66–134, and rA135–551 and then challenged by injection with live A. hydrophila. The development of the embryos was observed, and the cumulative mortality rate was calculated at 24 h after injection. OD, optical density. All data were expressed as means ± sd (n = 3). The data are from 3 independent experiments performed in triplicate. The bars represent the means ± sd. *P < 0.05, **P < 0.01.

Article Snippet: The PCR product was digested with Eco RI (Takara) and Hind III (Takara) and subcloned into the pET28a expression vector (Merck GmbH, Darmstadt, Germany) digested previously with the same restriction enzymes.

Techniques: SDS Page, Western Blot, Binding Assay, Marker, Negative Control, Plasmid Preparation, Expressing, Purification, Activity Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Injection

Journal: The FASEB Journal

Article Title: Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection

doi: 10.1096/fj.201901290R

Figure Lengend Snippet: SDS-PAGE and Western blotting of rMA1–65 and rCA1–65 and their antibacterial activities and binding to LTA and LPS. A) SDS-PAGE and Western blotting of recombinant protein rMA1–65 (a) and rCA1–65 (b). Lane M, marker; lane 1, negative control (empty vector pET28a); lane 2, total cellular extracts from E. coli BL21 containing expression vector pET28a/ma1-65 (a) and pET28a/ca1-65 (b) before induction; lane 3, total cellular extracts from IPTG-induced E. coli BL21 containing expression vector pET28a/ma1-65 (a) and pET28a/ca1-65 (b); lane 4, purified rMA1–65 (a) and rCA1–65 (b); lane 5, Western blotting of rMA1–65 (a) and rCA1–65 (b). B, C) Antibacterial activity of rMA1–65 (B) and rCA1–65 (C) against M. luteus, B. subtilis, S. aureus, E. coli, V. anguillarum, and A. hydrophila. D) Binding of rMA1–65 and rCA1–65 to LTA and LPS. OD, optical density. All data are expressed as means ± sd (n = 3). The data are from 3 independent experiments performed in triplicate. The bars represent means ± sd.

Article Snippet: The PCR product was digested with Eco RI (Takara) and Hind III (Takara) and subcloned into the pET28a expression vector (Merck GmbH, Darmstadt, Germany) digested previously with the same restriction enzymes.

Techniques: SDS Page, Western Blot, Binding Assay, Recombinant, Marker, Negative Control, Plasmid Preparation, Expressing, Purification, Activity Assay