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mbo388-sup-0001-FigS1.epsimage/eps11347KFigure S1. Purification of Ubiquitinated proteins and specific Lys48-polyUb proteins by immunoprecipitation. Cell Lysates of different encystation stages (0, 6, 12, 24, and 48 h) were prepared. Lysates (500 μg) supplemented with 10 mmol/L NEM and 20 μmol/L MG132 were incubated for 1 h with anti-Ub FK2 antibody or anti-Lys48-polyUb chain antibody. Immunoconjugates wre purified with protein A-SepharoseCL4B (GE HealthcareLife Sciences) and analyzed by immunoblotting with P4D1 anti-Ub monoclonal antibody. Fractions of different immunoprecipitation stages are indicated, Input: initial lysate, NB: not binding fraction, IP: immunoprecipitated, and LW: last wash.
mbo388-sup-0002-FigS2.epsimage/eps3546KFigure S2. Validation strategy, in vivo purification of ubiquitinated species from trophozoite extracts. Endogenous ubiquitinated proteins were purified by immunoprecipitation with anti-Ub FK2 antibody or anti-Lys48-polyUb chain antibody. (A) The ubiquitinated purified proteins were separated by SDS-PAGE and after staining the gel was cut and analyzed by HPLC-MS/MS. (B) A fraction of immunoprecipitated proteins was analyzed by immunobloting with P4D1 anti-Ub monoclonal antibody. Fractions of different immunoprecipitation stages are indicated, IP: immunoprecipitated, Input: initial lysate, NB: not binding fraction, and LW: last wash. (C) MS/MS spectrum of the linkage-specific peptide for Lys48 chains (signature peptide) with the Gly-Gly modified lysine residue. Peptide sequence is shown with the annotation of the identified matched b ions in red and the y ions in blue.
mbo388-sup-0003-FigS3.epsimage/eps3071KFigure S3. Sequence alignment of the HECT domains of human Nedd4 family members, E6AP, yeast R sp5, and two putative Giardia HECT (XP_001704118.1 and XP_0017006167.1), performed with Muscle. Sequences are color-coded according to sequence conservation. Uniprot accession codes qre provided with the protein names.
mbo388-sup-0004-TableS1.pdfapplication/PDF107KTable S1. Proteins identified with the in vitro ubiquitination assay.
mbo388-sup-0005-TableS2.pdfapplication/PDF99KTable S2. Proteins presents in both control and in vivo/in vitro ubiquitination assay.
mbo388-sup-0006-TableS3.pdfapplication/PDF89KTable S3. Sequence analysis of protein kinases, protein 21.1 and hypothetical proteins identofied with the in vivo/ in vitro ubiquitination assay.
mbo388-sup-0007-TableS4.pdfapplication/PDF53KTable S4. Ubiquitinations sites identified in the in vitro/in vivo analysis.
mbo388-sup-0008-TableS5.pdfapplication/PDF50KTable S5. Edogenous proteins from trophozoites purified by immunoprecipitation.

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