Journal: PLoS Pathogens

Article Title: Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

doi: 10.1371/journal.ppat.1002539

Figure Lengend Snippet: (A) Phagosome localization of CPBF8. Amoebae were incubated with Cell Tracker Blue-stained CHO cells ( blue ) for 10 (top row) or 60 minutes (bottom row), fixed, and reacted with anti-HA antibody ( green ). Bar , 10 µm. (B) Lysosomes localization of CPBF8. Amoebae were labeled with LysoTracker Red ( red ) and subjected to immunofluorescence assay with anti-HA antibody ( green ). Bar, 10 µm. (C) Colocalization of Eh PNT and CPBF8. The cells were fixed, and reacted with anti- Eh PNT ( red ) and anti-HA antibody ( green ). Bar, 10 µm.

Article Snippet: The expression plasmids for histidine-tagged CPBF8 (a.a.14–292) and lysozyme 2 were introduced into BL21(DE3) competent cells (Invitrogen).

Techniques: Incubation, Staining, Labeling, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

doi: 10.1371/journal.ppat.1002539

Figure Lengend Snippet: Lysates of CPBF8-HA and control (“HA”) transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained. Apparent molecular weight of standards (kDa) are indicated on the left. Six bands excised for protein identification are marked (A–F).

Article Snippet: The expression plasmids for histidine-tagged CPBF8 (a.a.14–292) and lysozyme 2 were introduced into BL21(DE3) competent cells (Invitrogen).

Techniques: Immunoprecipitation, SDS Page, Staining, Molecular Weight

Journal: PLoS Pathogens

Article Title: Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

doi: 10.1371/journal.ppat.1002539

Figure Lengend Snippet: Identification of CPBF-binding proteins by LC-MS/MS analysis.

Article Snippet: The expression plasmids for histidine-tagged CPBF8 (a.a.14–292) and lysozyme 2 were introduced into BL21(DE3) competent cells (Invitrogen).

Techniques:

Journal: PLoS Pathogens

Article Title: Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

doi: 10.1371/journal.ppat.1002539

Figure Lengend Snippet: (A) RT-PCR analysis. A 200 bp long partial CPBF8 gene was amplified using cDNA from control and CPBF8gs strains. (B) DNA microarray analysis of CPBF genes (CPBF1-11). The raw fluorescence data of triplicates is shown.

Article Snippet: The expression plasmids for histidine-tagged CPBF8 (a.a.14–292) and lysozyme 2 were introduced into BL21(DE3) competent cells (Invitrogen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Microarray, Fluorescence

Journal: PLoS Pathogens

Article Title: Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

doi: 10.1371/journal.ppat.1002539

Figure Lengend Snippet: (A) Schematic diagram of the serine-rich region and transmembrane domain in CPBF8. Numbers indicate amino acid positions from the amino terminus. The filled or hatched box depicts the serine rich region or the transmembrane domain, respectively. (B) Comparison of the carboxyl-terminal region of CPBF proteins. Boxes indicate the putative transmembrane domain. The serine-rich region is underlined. (C) The amino acid sequences of the wild-type and mutated serine-rich regions (SRR). Note that the entire SRR was deleted in CPBF8ΔSRR-HA. The first or second stretch of three serine residues within SRR were substituted with alanines in CPBF8AAA1-HA and CPBF8AAA2-HA, respectively. (D) Localization of CPBF8ΔSRR-HA to phagosomes. Amoebae were incubated with Cell Tracker Blue-stained CHO cells ( blue ) for 60 min, fixed, and reacted with anti-HA antibody ( green ). Bar, 10 µm. (E–F) Isolation and identification of binding proteins of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA. Lysates of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained (The upper and lower arrow indicated that β-hexosaminidase α-subunit and lysozymes, respectively. (E), or blotted and reacted with anti-HA, β-hexosaminidase α-subunit and lysozyme2 antibody (F).

Article Snippet: The expression plasmids for histidine-tagged CPBF8 (a.a.14–292) and lysozyme 2 were introduced into BL21(DE3) competent cells (Invitrogen).

Techniques: Incubation, Staining, Isolation, Binding Assay, Immunoprecipitation, SDS Page

Journal: PLoS Pathogens

Article Title: Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

doi: 10.1371/journal.ppat.1002539

Figure Lengend Snippet: CPBF8-HA and CPBF8ΔSRR-HA was immunoprecipitated with anti-HA antibody from the lysates of CPBF8-HA and CPBF8ΔSRR-HA transformants, treated (+) or untreated (−) with TFA for 10 min. The samples were separated on SDS-PAGE and silver-stained (bottom panel), or blotted and reacted with anti-HA (top panel) or anti-lysozyme 2 antibody (middle panel). The apparent molecular weight (kDa) of standards are indicated on the left.

Article Snippet: The expression plasmids for histidine-tagged CPBF8 (a.a.14–292) and lysozyme 2 were introduced into BL21(DE3) competent cells (Invitrogen).

Techniques: Immunoprecipitation, SDS Page, Staining, Molecular Weight