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  • 99
    Millipore anti ha antibodies
    Effect of pathogenic CDKL5 mutations on CDKL5 activity HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and HA‐tagged MAP1S. <t>Anti‐HA</t> precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the MAP1S Ser 900 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged CEP131. Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the CEP131 Ser 35 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.
    Anti Ha Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore murine antiha antibody
    Effect of pathogenic CDKL5 mutations on CDKL5 activity HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and HA‐tagged MAP1S. <t>Anti‐HA</t> precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the MAP1S Ser 900 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged CEP131. Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the CEP131 Ser 35 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.
    Murine Antiha Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore rabbit anti ha antibodies
    Effect of pathogenic CDKL5 mutations on CDKL5 activity HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and HA‐tagged MAP1S. <t>Anti‐HA</t> precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the MAP1S Ser 900 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged CEP131. Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the CEP131 Ser 35 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.
    Rabbit Anti Ha Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse anti ha antibody
    Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using <t>anti‐HA/Strep/FLAG</t> antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).
    Mouse Anti Ha Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 533 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti ha antibody
    Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using <t>anti‐HA/Strep/FLAG</t> antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).
    Anti Ha Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 931 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore anti ha tag antibody
    CXCR4-[P2G]CXCL12 intermolecular crosslinking efficiencies detected by non-reducing SDS PAGE and Western blotting of pulled down complexes. ( A ) Non-reducing SDS-PAGE and Western blot of His-pulldowns of select combinations of CXCR4 and [P2G]CXCL12 cysteine mutants. The Flag-CXCR4-T4L receptor and <t>[P2G]CXCL12-HA</t> chemokine were detected by LI-COR IRDye ® conjugated secondary <t>antibodies</t> on a single blot (emission wavelength of 680 nm and 800 nm, visualized in red and green, respectively). Emitted fluorescence detected at 800 nm and 680 nm from the same band of the Western blot is indicative of the receptor and chemokine co-migrating on the gel and thus crosslinked. ( B ) Comparison of crosslinking efficiency determined by flow cytometry (black bars) and Western blotting (green bars). Crosslinking efficiency by Western blotting is given by the <t>anti-HA:IR800</t> fluorescence intensity. ( C ) The percentage of crosslinked receptor obtained from Western blotting of the pull-down samples, calculated as the ratio of the IR680 (red) signal intensity of the upper Flag band to the total receptor IR680 signal. Data is representative of n = 3 independent replicates. The mean and s.e.m are reported for each point.
    Anti Ha Tag Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Boehringer Mannheim anti ha ab
    CXCR4-[P2G]CXCL12 intermolecular crosslinking efficiencies detected by non-reducing SDS PAGE and Western blotting of pulled down complexes. ( A ) Non-reducing SDS-PAGE and Western blot of His-pulldowns of select combinations of CXCR4 and [P2G]CXCL12 cysteine mutants. The Flag-CXCR4-T4L receptor and <t>[P2G]CXCL12-HA</t> chemokine were detected by LI-COR IRDye ® conjugated secondary <t>antibodies</t> on a single blot (emission wavelength of 680 nm and 800 nm, visualized in red and green, respectively). Emitted fluorescence detected at 800 nm and 680 nm from the same band of the Western blot is indicative of the receptor and chemokine co-migrating on the gel and thus crosslinked. ( B ) Comparison of crosslinking efficiency determined by flow cytometry (black bars) and Western blotting (green bars). Crosslinking efficiency by Western blotting is given by the <t>anti-HA:IR800</t> fluorescence intensity. ( C ) The percentage of crosslinked receptor obtained from Western blotting of the pull-down samples, calculated as the ratio of the IR680 (red) signal intensity of the upper Flag band to the total receptor IR680 signal. Data is representative of n = 3 independent replicates. The mean and s.e.m are reported for each point.
    Anti Ha Ab, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche anti ha ab
    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using <t>anti‐HA</t> antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
    Anti Ha Ab, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti ha antibody
    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using <t>anti‐HA</t> antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
    Anti Ha Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche monoclonal anti ha antibody
    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using <t>anti‐HA</t> antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
    Monoclonal Anti Ha Antibody, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of pathogenic CDKL5 mutations on CDKL5 activity HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and HA‐tagged MAP1S. Anti‐HA precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the MAP1S Ser 900 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged CEP131. Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the CEP131 Ser 35 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase

    doi: 10.15252/embj.201899559

    Figure Lengend Snippet: Effect of pathogenic CDKL5 mutations on CDKL5 activity HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and HA‐tagged MAP1S. Anti‐HA precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the MAP1S Ser 900 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged CEP131. Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Hi, high exposure; lo, low exposure. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the CEP131 Ser 35 phosphorylation site, in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ . Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Article Snippet: Anti‐FLAG (M2) and anti‐HA antibodies were obtained from Sigma, and anti‐GAPDH (14C10) antibodies were purchased from Cell Signalling.

    Techniques: Activity Assay, Transfection, Western Blot, Expressing, Incubation, Sequencing

    CDKLs 1–4 cannot phosphorylate MAP1S or CEP131 in cells Schematic representation of CDKLs 1–5. The kinase catalytic domain is highlighted in dark blue. Amino acid numbers at the N‐ and C‐termini are indicated; the positions of conserved residues in the ATP binding sites that were mutated to render these protein “kinase‐dead” are also indicated. HEK293 cells were co‐transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant) and HA‐MAP1S. Anti‐HA precipitates were subjected to Western blotting with the antibodies indicated. “Hi” higher exposure; “lo” lower exposure. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Same as (B) except that HEK293 cells were co‐transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant) and HA‐CEP131. Three independent experiments were done, and one representative experiment is shown. HEK293 cells were transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant). Anti‐FLAG precipitates were incubated with the MAP1S S 900 synthetic peptide in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ , and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase

    doi: 10.15252/embj.201899559

    Figure Lengend Snippet: CDKLs 1–4 cannot phosphorylate MAP1S or CEP131 in cells Schematic representation of CDKLs 1–5. The kinase catalytic domain is highlighted in dark blue. Amino acid numbers at the N‐ and C‐termini are indicated; the positions of conserved residues in the ATP binding sites that were mutated to render these protein “kinase‐dead” are also indicated. HEK293 cells were co‐transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant) and HA‐MAP1S. Anti‐HA precipitates were subjected to Western blotting with the antibodies indicated. “Hi” higher exposure; “lo” lower exposure. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown. Same as (B) except that HEK293 cells were co‐transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant) and HA‐CEP131. Three independent experiments were done, and one representative experiment is shown. HEK293 cells were transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant). Anti‐FLAG precipitates were incubated with the MAP1S S 900 synthetic peptide in the presence of [γ‐ 32 P]‐labelled ATP‐Mg 2+ , and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Article Snippet: Anti‐FLAG (M2) and anti‐HA antibodies were obtained from Sigma, and anti‐GAPDH (14C10) antibodies were purchased from Cell Signalling.

    Techniques: Binding Assay, Transfection, Mutagenesis, Western Blot, Incubation

    Distribution of DDX3 and IPS-1. (A) DDX3 colocalizes with IPS-1 on the mitochondria in Oc cells. HA-tagged DDX3 and FLAG-tagged IPS-1 were co-transfected into Oc cells. After 24 hrs, cells were fixed with formaldehyde and stained with anti-HA polyclonal and FLAG monoclonal Abs. Alexa488 (DDX3-HA) or Alexa633 antibody was used for second antibody. Mitochondria were stained with Mitotracker Red. Similar IPS-1-DDX3 merging profiles were observed in Huh7.5.1 cells ( Fig. S3 ). (B,C) O cells with the HCV replicon poorly formed the DDX3-IPS-1 complex. Plasmids carrying IPS-1 (100 ng) or DDX3 (150 or 300 ng) were transfected into O (HCV replicon +) as in Oc cells (no replicon, panel A). After 24 hrs, localization of IPS-1 and DDX3 was examined by confocal microscopy. Two representatives which differ from the conventional profile (as in panel A) are shown. Similar sets of experiments were performed four times to confirm the results.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Core Protein Abrogates the DDX3 Function That Enhances IPS-1-Mediated IFN-Beta Induction

    doi: 10.1371/journal.pone.0014258

    Figure Lengend Snippet: Distribution of DDX3 and IPS-1. (A) DDX3 colocalizes with IPS-1 on the mitochondria in Oc cells. HA-tagged DDX3 and FLAG-tagged IPS-1 were co-transfected into Oc cells. After 24 hrs, cells were fixed with formaldehyde and stained with anti-HA polyclonal and FLAG monoclonal Abs. Alexa488 (DDX3-HA) or Alexa633 antibody was used for second antibody. Mitochondria were stained with Mitotracker Red. Similar IPS-1-DDX3 merging profiles were observed in Huh7.5.1 cells ( Fig. S3 ). (B,C) O cells with the HCV replicon poorly formed the DDX3-IPS-1 complex. Plasmids carrying IPS-1 (100 ng) or DDX3 (150 or 300 ng) were transfected into O (HCV replicon +) as in Oc cells (no replicon, panel A). After 24 hrs, localization of IPS-1 and DDX3 was examined by confocal microscopy. Two representatives which differ from the conventional profile (as in panel A) are shown. Similar sets of experiments were performed four times to confirm the results.

    Article Snippet: Anti-FLAG M2 monoclonal Ab and anti-HA polyclonal Ab were purchased from Sigma.

    Techniques: Transfection, Staining, Confocal Microscopy

    Environmental [K + ]-induced dephosphorylation of ACBP2 is associated with its translocation. (A) Western blot analysis of ACBP2 expression in extracellular and intracellular tachyzoites. Intracellular and extracellular tachyzoites were harvested and incubated in IC buffer and EC buffer for 2 h, respectively. Additionally, extracellular tachyzoites were incubated in IC buffer for 2 h. Two forms of ACBP2 were present in extracellular and intracellular tachyzoites. Extracellular tachyzoites underwent stimulation by IC buffer and produced a smaller band consistent with the size of intracellular ACBP2. Mouse anti-ACBP2 antibody was used to detect the expression of ACBP2. Actin1 was used as the loading control. EC, extracellular tachyzoites; IC, intracellular tachyzoites. (B) Alkaline phosphatase (AP) assay for the phosphorylation of TgACBP2. Endogenous TgACBP2 immunoprecipitated (IP) from both intracellular and extracellular TgACBP2-HA parasites using antibodies against HA was incubated with or without AP for 0.5 h and 1 h; ACBP2 from extracellular tachyzoites began to be dephosphorylated (upper panel) and was completely dephosphorylated after 1 h (lower panel). “pACBP2” represents “phosphorylated ACBP2.” Mouse anti-ACBP2 antibody was used to detect the expression of ACBP2. (C) Extracellular TgACBP2-HA tachyzoites incubated with 25 mM, 125 mM, and 300 mM KCl and NaCl buffers for 2 h were analyzed by Western blotting using antibodies against HA. [K + ] induced ACBP2 dephosphorylation in a concentration-dependent manner. Anti-HA was used to detect the expression of ACBP2. Actin1 was used as the loading control. (D) Extracellular TgACBP2-HA parasites were harvested and incubated with IC buffer for 2 h. IFA showed that IC buffer induced the translocation of TgACBP2. Anti-HA was used to detect the localization of ACBP2. Bar, 5 μm. (E) The association of [Ca 2+ ] with TgACBP2 dephosphorylation was assessed using 1% ethanol and Ca 2+ chelator BAPTA-AM. Extracellular TgACBP2-HA parasites were harvested and incubated in EC buffer and IC buffer. After the addition of 1% ethanol to the IC buffer, extracellular parasites in IC buffer were not dephosphorylated, while treatment of extracellular parasites with BAPTA-AM in EC buffer induced ACBP2 dephosphorylation. Anti-HA was used to detect the expression of ACBP2. Actin1 was used as the loading control.

    Journal: mBio

    Article Title: Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism

    doi: 10.1128/mBio.01597-18

    Figure Lengend Snippet: Environmental [K + ]-induced dephosphorylation of ACBP2 is associated with its translocation. (A) Western blot analysis of ACBP2 expression in extracellular and intracellular tachyzoites. Intracellular and extracellular tachyzoites were harvested and incubated in IC buffer and EC buffer for 2 h, respectively. Additionally, extracellular tachyzoites were incubated in IC buffer for 2 h. Two forms of ACBP2 were present in extracellular and intracellular tachyzoites. Extracellular tachyzoites underwent stimulation by IC buffer and produced a smaller band consistent with the size of intracellular ACBP2. Mouse anti-ACBP2 antibody was used to detect the expression of ACBP2. Actin1 was used as the loading control. EC, extracellular tachyzoites; IC, intracellular tachyzoites. (B) Alkaline phosphatase (AP) assay for the phosphorylation of TgACBP2. Endogenous TgACBP2 immunoprecipitated (IP) from both intracellular and extracellular TgACBP2-HA parasites using antibodies against HA was incubated with or without AP for 0.5 h and 1 h; ACBP2 from extracellular tachyzoites began to be dephosphorylated (upper panel) and was completely dephosphorylated after 1 h (lower panel). “pACBP2” represents “phosphorylated ACBP2.” Mouse anti-ACBP2 antibody was used to detect the expression of ACBP2. (C) Extracellular TgACBP2-HA tachyzoites incubated with 25 mM, 125 mM, and 300 mM KCl and NaCl buffers for 2 h were analyzed by Western blotting using antibodies against HA. [K + ] induced ACBP2 dephosphorylation in a concentration-dependent manner. Anti-HA was used to detect the expression of ACBP2. Actin1 was used as the loading control. (D) Extracellular TgACBP2-HA parasites were harvested and incubated with IC buffer for 2 h. IFA showed that IC buffer induced the translocation of TgACBP2. Anti-HA was used to detect the localization of ACBP2. Bar, 5 μm. (E) The association of [Ca 2+ ] with TgACBP2 dephosphorylation was assessed using 1% ethanol and Ca 2+ chelator BAPTA-AM. Extracellular TgACBP2-HA parasites were harvested and incubated in EC buffer and IC buffer. After the addition of 1% ethanol to the IC buffer, extracellular parasites in IC buffer were not dephosphorylated, while treatment of extracellular parasites with BAPTA-AM in EC buffer induced ACBP2 dephosphorylation. Anti-HA was used to detect the expression of ACBP2. Actin1 was used as the loading control.

    Article Snippet: For antibodies (Abs), mouse anti-HA monoclonal Ab (MAb) was purchased from Sigma.

    Techniques: De-Phosphorylation Assay, Translocation Assay, Western Blot, Expressing, Incubation, Produced, Immunoprecipitation, Concentration Assay, Immunofluorescence

    Localization of TgACBP2 in extracellular and intracellular parasites. (A) Western blot analysis of TgACBP2-HA produced by endogenous C-terminal tagging. Parasite lysates were probed with anti-HA (upper panel); Actin1 served as the loading control (lower panel). (B) Localization of TgACBP2 was further determined by IFA using two different mitochondrial markers, F 1 bATPase and MitoTrackerRed CMXRos. Anti-HA was used to label TgACBP2, and SAG1 was used as a parasite membrane marker. Bars = 5 μm. (C) The solubility of TgACBP2 was investigated by fractionation experiments after the ultrasonication of tachyzoites in PBS, NaCl, and Na 2 CO 3 solutions. The distribution of TgACBP2 fractions was measured by Western blotting using mouse anti-TgACBP2 antibody; Actin1 and Profilin were used as controls for membrane-associated and soluble proteins, respectively. (D) A proteinase K protection assay was used to determine the accessibility of ACBP2 to proteinase K in the presence or absence of the detergent Triton X-100 (TX). After permeabilization by TX, PK could easily pass through organelle membranes to stroma and digest stoma proteins nonspecifically. For ACBP2, even without TX, PK was also able to degrade ACBP2, further indicating that ACBP2 is localized on the membrane of mitochondria. Actin1 served as a marker of cytosolic proteins, while ACP and cytochrome c (CytC) served as markers of apicoplast stroma and inner mitochondria, respectively. Mouse anti-ACBP2 antibody was used to detect the expression of ACBP2. (E) The subcellular localization of ACBP2 was determined in both intracellular and extracellular tachyzoites endogenously tagged with TgACBP2-HA. Anti-HA was used to detect the distribution of ACBP2, and SAG1 was used as a membrane marker. ACBP2 was localized to mitochondria in intracellular tachyzoites and yet translocated to the plasma membrane in extracellular tachyzoites. Bars = 5 μm.

    Journal: mBio

    Article Title: Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism

    doi: 10.1128/mBio.01597-18

    Figure Lengend Snippet: Localization of TgACBP2 in extracellular and intracellular parasites. (A) Western blot analysis of TgACBP2-HA produced by endogenous C-terminal tagging. Parasite lysates were probed with anti-HA (upper panel); Actin1 served as the loading control (lower panel). (B) Localization of TgACBP2 was further determined by IFA using two different mitochondrial markers, F 1 bATPase and MitoTrackerRed CMXRos. Anti-HA was used to label TgACBP2, and SAG1 was used as a parasite membrane marker. Bars = 5 μm. (C) The solubility of TgACBP2 was investigated by fractionation experiments after the ultrasonication of tachyzoites in PBS, NaCl, and Na 2 CO 3 solutions. The distribution of TgACBP2 fractions was measured by Western blotting using mouse anti-TgACBP2 antibody; Actin1 and Profilin were used as controls for membrane-associated and soluble proteins, respectively. (D) A proteinase K protection assay was used to determine the accessibility of ACBP2 to proteinase K in the presence or absence of the detergent Triton X-100 (TX). After permeabilization by TX, PK could easily pass through organelle membranes to stroma and digest stoma proteins nonspecifically. For ACBP2, even without TX, PK was also able to degrade ACBP2, further indicating that ACBP2 is localized on the membrane of mitochondria. Actin1 served as a marker of cytosolic proteins, while ACP and cytochrome c (CytC) served as markers of apicoplast stroma and inner mitochondria, respectively. Mouse anti-ACBP2 antibody was used to detect the expression of ACBP2. (E) The subcellular localization of ACBP2 was determined in both intracellular and extracellular tachyzoites endogenously tagged with TgACBP2-HA. Anti-HA was used to detect the distribution of ACBP2, and SAG1 was used as a membrane marker. ACBP2 was localized to mitochondria in intracellular tachyzoites and yet translocated to the plasma membrane in extracellular tachyzoites. Bars = 5 μm.

    Article Snippet: For antibodies (Abs), mouse anti-HA monoclonal Ab (MAb) was purchased from Sigma.

    Techniques: Western Blot, Produced, Immunofluorescence, Marker, Solubility, Fractionation, Expressing

    MAF1-mediated HMA rescued the growth defect of PruΔ acbp2 tachyzoites. (A) Construction of PruΔ acbp2 tachyzoites expressing an N-terminally HA-tagged MAF1RHb1 was confirmed by Western blot analysis using anti-HA and mouse anti-ACBP2 antibody. Actin1 was used as the loading control. (B) Expression of MAF1RHb1 in Pru parasites was further confirmed by IFA using antibodies against HA and SAG1. MAF1RHb1 is localized on the PVM. Bar, 5 μm. (C) MAF1RHb1-mediated HMA was assessed by IFA using MitoTracker. MAF1 could recruit host mitochondria around the Toxoplasma PVM in PruΔ acbp2 /MAF1RHb1 parasites. Bar, 5 μm. (D) Proliferation assay showed that MAF1RHb1 expression in ACBP2-deficient parasites fully complemented the replication defect. The values are representative of the means ± SEM of results from three independent experiments. (E and F) Plaque assays were performed by infecting HFFs with 100 Pru, PruΔ acbp2 , or PruΔ acbp2 /MAF1RHb1 parasites and cultivating them for 12 days (E). Plaque area analysis demonstrated that MAF1RHb1 expression rescued the growth defects of the PruΔ acbp2 parasites (F). The values are representative of the means ± SEM of results from two experiments. (G) Apoptosis of Pru, PruΔ acbp2 , and PruΔ acbp2 /MAF1RHb1 tachyzoites was assessed by the TUNEL assay. The results showed that MAF1RHb1 expression rescued the apoptosis defect caused by loss of ACBP2 in Pru parasites. Data are presented as the means ± SEM of results from three independent experiments. (H) ΔΨm was determined in Pru, PruΔ acbp2 , and PruΔ acbp2 /MAF1RHb1 tachyzoites by DiOC6(3) staining. FACS results showed that MAF1RHb1 expression rescued the decresed mitochondrial membrane potential caused by disruption of ACBP2 in Pru parasites. The error bars represent the standard errors of the means of results from three independent experiments.

    Journal: mBio

    Article Title: Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism

    doi: 10.1128/mBio.01597-18

    Figure Lengend Snippet: MAF1-mediated HMA rescued the growth defect of PruΔ acbp2 tachyzoites. (A) Construction of PruΔ acbp2 tachyzoites expressing an N-terminally HA-tagged MAF1RHb1 was confirmed by Western blot analysis using anti-HA and mouse anti-ACBP2 antibody. Actin1 was used as the loading control. (B) Expression of MAF1RHb1 in Pru parasites was further confirmed by IFA using antibodies against HA and SAG1. MAF1RHb1 is localized on the PVM. Bar, 5 μm. (C) MAF1RHb1-mediated HMA was assessed by IFA using MitoTracker. MAF1 could recruit host mitochondria around the Toxoplasma PVM in PruΔ acbp2 /MAF1RHb1 parasites. Bar, 5 μm. (D) Proliferation assay showed that MAF1RHb1 expression in ACBP2-deficient parasites fully complemented the replication defect. The values are representative of the means ± SEM of results from three independent experiments. (E and F) Plaque assays were performed by infecting HFFs with 100 Pru, PruΔ acbp2 , or PruΔ acbp2 /MAF1RHb1 parasites and cultivating them for 12 days (E). Plaque area analysis demonstrated that MAF1RHb1 expression rescued the growth defects of the PruΔ acbp2 parasites (F). The values are representative of the means ± SEM of results from two experiments. (G) Apoptosis of Pru, PruΔ acbp2 , and PruΔ acbp2 /MAF1RHb1 tachyzoites was assessed by the TUNEL assay. The results showed that MAF1RHb1 expression rescued the apoptosis defect caused by loss of ACBP2 in Pru parasites. Data are presented as the means ± SEM of results from three independent experiments. (H) ΔΨm was determined in Pru, PruΔ acbp2 , and PruΔ acbp2 /MAF1RHb1 tachyzoites by DiOC6(3) staining. FACS results showed that MAF1RHb1 expression rescued the decresed mitochondrial membrane potential caused by disruption of ACBP2 in Pru parasites. The error bars represent the standard errors of the means of results from three independent experiments.

    Article Snippet: For antibodies (Abs), mouse anti-HA monoclonal Ab (MAb) was purchased from Sigma.

    Techniques: Expressing, Western Blot, Immunofluorescence, Proliferation Assay, TUNEL Assay, Staining, FACS

    STAT3 mono-ubiquitination in cellulo . (A) HEK 293 cells were transfected either with V5- tagged STAT3 alone (lane 1 and 2) or along with HA- tagged WT ubi (lane 3) or HA- tagged K0 ubi (lane 4). 40 h after transfection, cells were treated with 1.5 μM MG132 for 4 h prior to 15 min stimulation with IL-6 (8ng/ml) and sIL-6R (6 μg/ml). Cells were harvested with modified RIPA and WCL were IP’ed either with IgG (lane 1) or with anti-V5 Ab (lane 2, 3, 4) and immunoblotted with anti-HA Ab (top) or with anti-V5 Ab (middle). The bottom panel shows input samples (5% WCL without IP) subjected to western immunoblot analysis wih anti-STAT3 Ab. (B) HEK 293 cells were transfected with V5-STAT3 and HA-ubi-K0 and WCL were IP’ed either with IgG (lane 1) or with anti-HA Ab from mouse (M) (lane 2) or anti-HA Ab from rabbit (R) (lane 3) or with anti-STAT3 Ab (lane 4) and Western immunoblot was performed with anti-V5 Ab. (C) Cells were transfected with either V5-His-STAT3 alone (lane 1) or with V5-His-STAT3 and HA-ubi-WT (lane 2) or V5-His-STAT3 and HA-ubi-K0 (lane 3). WCL were subjected to Ni-affinity NTA pull down assay. The eluted fraction were resolved in SDS-PAGE and immunoblotted with anti-HA Ab (top) and anti-V5 Ab (bottom). (D) 293 cells were transfected either with ubi-K0 (lane 1and 2) or ubi-WT (lane 3) expression plasmid and WCL were IPed with anti-STAT3 Ab and immunoblotted with either anti-STAT3 Ab (left) or with anti-ubi Ab (right). The bottom left panel is lane 1 after longer exposure, showing ubiquitin modified STAT3. (E) Cells were transfected with V5-His-STAT3 and HA-ubi-K0 and prior to harvest, cells were stimulated either with IL-6 or with H 2 O 2 or left untreated. WCL were pull down by Ni-NTA assay and eluted proteins were resolved and immunoblotted with anti-HA Ab. Shown is the average relative intensity of ubi-STAT3 expression (pulled down in Ni-NTA assay (n=3)) in the Western Blot. #, represents p

    Journal: Cellular signalling

    Article Title: Inducible STAT3 NH2 Terminal Mono-ubiquitination Promotes BRD4 Complex Formation to Regulate Apoptosis

    doi: 10.1016/j.cellsig.2014.03.007

    Figure Lengend Snippet: STAT3 mono-ubiquitination in cellulo . (A) HEK 293 cells were transfected either with V5- tagged STAT3 alone (lane 1 and 2) or along with HA- tagged WT ubi (lane 3) or HA- tagged K0 ubi (lane 4). 40 h after transfection, cells were treated with 1.5 μM MG132 for 4 h prior to 15 min stimulation with IL-6 (8ng/ml) and sIL-6R (6 μg/ml). Cells were harvested with modified RIPA and WCL were IP’ed either with IgG (lane 1) or with anti-V5 Ab (lane 2, 3, 4) and immunoblotted with anti-HA Ab (top) or with anti-V5 Ab (middle). The bottom panel shows input samples (5% WCL without IP) subjected to western immunoblot analysis wih anti-STAT3 Ab. (B) HEK 293 cells were transfected with V5-STAT3 and HA-ubi-K0 and WCL were IP’ed either with IgG (lane 1) or with anti-HA Ab from mouse (M) (lane 2) or anti-HA Ab from rabbit (R) (lane 3) or with anti-STAT3 Ab (lane 4) and Western immunoblot was performed with anti-V5 Ab. (C) Cells were transfected with either V5-His-STAT3 alone (lane 1) or with V5-His-STAT3 and HA-ubi-WT (lane 2) or V5-His-STAT3 and HA-ubi-K0 (lane 3). WCL were subjected to Ni-affinity NTA pull down assay. The eluted fraction were resolved in SDS-PAGE and immunoblotted with anti-HA Ab (top) and anti-V5 Ab (bottom). (D) 293 cells were transfected either with ubi-K0 (lane 1and 2) or ubi-WT (lane 3) expression plasmid and WCL were IPed with anti-STAT3 Ab and immunoblotted with either anti-STAT3 Ab (left) or with anti-ubi Ab (right). The bottom left panel is lane 1 after longer exposure, showing ubiquitin modified STAT3. (E) Cells were transfected with V5-His-STAT3 and HA-ubi-K0 and prior to harvest, cells were stimulated either with IL-6 or with H 2 O 2 or left untreated. WCL were pull down by Ni-NTA assay and eluted proteins were resolved and immunoblotted with anti-HA Ab. Shown is the average relative intensity of ubi-STAT3 expression (pulled down in Ni-NTA assay (n=3)) in the Western Blot. #, represents p

    Article Snippet: Sources of primary antibody (Ab) included Santa Cruz Biotechnology (Dallas TX) for anti-STAT3 Ab (C20) and anti-HA Ab; anti-V5 Ab was from Invitrogen (Grand Island, NY); anti β-actin Ab, anti-Flag M2Ab, Flag agarose beads and 3X Flag peptides were from Sigma-Aldrich (St. Louis, MO); anti- mouse monoclonal anti-HA Ab was from Millipore Billerica, MA).

    Techniques: Transfection, Modification, Western Blot, Pull Down Assay, SDS Page, Expressing, Plasmid Preparation

    Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using anti‐HA/Strep/FLAG antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using anti‐HA/Strep/FLAG antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Western Blot, SDS Page, Blue Native PAGE, Polymerase Chain Reaction, Staining, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Mitochondrial localisation of putative mitochondrial ribosomal proteins by epitope tagging. A. Localisation through fluorescence microscopy analysis of 4 gene‐products predicted to encode for mitoribosomal proteins identified in our search. B. Localisation of 2 additional gene‐products predicted to be mitochondrial ribosomal proteins not found in our search. For both panels, mitochondria are marked with anti‐TgMys or anti‐TOM40; the mentioned GOI (genes of interest) are marked with anti‐HA, anti‐FLAG or anti‐Strep. Scale bar 1 μm. Images in A also appear in Fig. S2 . [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Mitochondrial localisation of putative mitochondrial ribosomal proteins by epitope tagging. A. Localisation through fluorescence microscopy analysis of 4 gene‐products predicted to encode for mitoribosomal proteins identified in our search. B. Localisation of 2 additional gene‐products predicted to be mitochondrial ribosomal proteins not found in our search. For both panels, mitochondria are marked with anti‐TgMys or anti‐TOM40; the mentioned GOI (genes of interest) are marked with anti‐HA, anti‐FLAG or anti‐Strep. Scale bar 1 μm. Images in A also appear in Fig. S2 . [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Fluorescence, Microscopy

    CXCR4-[P2G]CXCL12 intermolecular crosslinking efficiencies detected by non-reducing SDS PAGE and Western blotting of pulled down complexes. ( A ) Non-reducing SDS-PAGE and Western blot of His-pulldowns of select combinations of CXCR4 and [P2G]CXCL12 cysteine mutants. The Flag-CXCR4-T4L receptor and [P2G]CXCL12-HA chemokine were detected by LI-COR IRDye ® conjugated secondary antibodies on a single blot (emission wavelength of 680 nm and 800 nm, visualized in red and green, respectively). Emitted fluorescence detected at 800 nm and 680 nm from the same band of the Western blot is indicative of the receptor and chemokine co-migrating on the gel and thus crosslinked. ( B ) Comparison of crosslinking efficiency determined by flow cytometry (black bars) and Western blotting (green bars). Crosslinking efficiency by Western blotting is given by the anti-HA:IR800 fluorescence intensity. ( C ) The percentage of crosslinked receptor obtained from Western blotting of the pull-down samples, calculated as the ratio of the IR680 (red) signal intensity of the upper Flag band to the total receptor IR680 signal. Data is representative of n = 3 independent replicates. The mean and s.e.m are reported for each point.

    Journal: bioRxiv

    Article Title: Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

    doi: 10.1101/2020.01.10.900951

    Figure Lengend Snippet: CXCR4-[P2G]CXCL12 intermolecular crosslinking efficiencies detected by non-reducing SDS PAGE and Western blotting of pulled down complexes. ( A ) Non-reducing SDS-PAGE and Western blot of His-pulldowns of select combinations of CXCR4 and [P2G]CXCL12 cysteine mutants. The Flag-CXCR4-T4L receptor and [P2G]CXCL12-HA chemokine were detected by LI-COR IRDye ® conjugated secondary antibodies on a single blot (emission wavelength of 680 nm and 800 nm, visualized in red and green, respectively). Emitted fluorescence detected at 800 nm and 680 nm from the same band of the Western blot is indicative of the receptor and chemokine co-migrating on the gel and thus crosslinked. ( B ) Comparison of crosslinking efficiency determined by flow cytometry (black bars) and Western blotting (green bars). Crosslinking efficiency by Western blotting is given by the anti-HA:IR800 fluorescence intensity. ( C ) The percentage of crosslinked receptor obtained from Western blotting of the pull-down samples, calculated as the ratio of the IR680 (red) signal intensity of the upper Flag band to the total receptor IR680 signal. Data is representative of n = 3 independent replicates. The mean and s.e.m are reported for each point.

    Article Snippet: HA-tagged [P2G]CXCL12 was detected using a rabbit anti-HA primary antibody (1:10,000 dilution; Sigma Aldrich, catalog #SAB4300603) and IRDye® 800CW-conjugated goat anti-rabbit IgG (1:20,000 dilution; LI-COR Biosciences, catalog #926-32211) secondary antibody.

    Techniques: SDS Page, Western Blot, Fluorescence, Flow Cytometry

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    doi: 10.15252/embr.201846331

    Figure Lengend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Article Snippet: Antibodies and immunoblotting The following commercial antibodies (Ab) were used for immunoblotting assays: Anti‐histone H3 C‐terminal Ab (Abcam Ab1791) were used at a 1:150,000 dilution, anti‐histone H3 N‐terminal Ab (Sigma‐9289) were used at 1:1,000 dilution, anti‐histone H4 Ab (Santa Cruz sc‐8658‐R) were used at 1:3,000 dilution, anti‐H3K9me3 Ab (Millipore 07‐442) were used at a 1:3,000 dilution, anti‐H3K18ac Ab (Millipore 07‐354) were used at a 1:40,000 dilution, anti‐H3K23ac Ab (Abcam ab46982) were used at a 1:100,000 dilution, anti‐H3K27me3 Ab (Abcam ab6147) were used at a 1:2,000, anti‐H3K27ac Ab (Millipore 07‐360) were used at a 1:40,000 dilution, anti‐GST Ab (Invitrogen 13‐6700) were used at a 1:50,000 dilution, and anti‐HA Ab (Roche 12CA5) were used at a 1:2,000 dilution.

    Techniques: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection