Journal: Genes & Development

Article Title: Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5

doi: 10.1101/gad.252676.114

Figure Lengend Snippet: Activation of the Hh pathway in LAP-Smo-expressing MEFs induces the interaction of Smo and Dlg5. ( A ) Gli -luciferase activity in LAP-Smo MEFs treated with ShhN conditioned medium, SAG1.5, and/or cyclopamine. ( B ) Microscopy analysis of untreated or ShhN-stimulated LAP-Smo MEFs stained using antibodies against acetylated tubulin (red) to mark the primary cilium and GFP (green) to detect LAP-Smo. The boxed region is magnified and shown as a shifted overlay demonstrating colocalization of LAP-Smo and acetylated tubulin. The fraction of primary cilia that overlaps with Smo is quantified and plotted. All error bars are SD. n > 50 cilia. ( C ) A schematic representation of the SILAC-based labeling of LAP-Smo MEFs for the identification of Smo-interacting proteins by LC−MS/MS. ( D ) A scatter plot depicting results of the quantitative comparison of proteins binding to LAP-Smo in Shh/SAG1.5-stimulated and untreated MEFs. The logarithmic ratios of protein abundance are plotted against the number of unique peptides detected for each protein. The position of Smo on the graph is denoted by a star symbol in blue. ShhNp/SAG treatment significantly enriches the amount of Dlg5 binding to Smo. ( E ) Coimmunoprecipitation with an anti-GFP matrix of lysates from HEK293T cells transfected with LAP-Smo and Dlg5-3xFlag, demonstrating specific binding of Dlg5 and Smo. ( F ) Coimmunoprecipitation with an anti-GFP matrix of lysates from LAP-Smo2 MEFs stimulated with ShhN conditioned medium for the indicated times and incubated with 1 mM DSP cross-linker. Shh stimulation increases the amount of Dlg5 bound to Smo. (*) Nonspecific bands.

Article Snippet: SAG1.5 was purchased from Enzo Life Sciences, DDM and CHS were purchased from Affymetrix, and Cytochalasin B and nocodazole were purchased from Sigma.

Techniques: Activation Assay, Expressing, Luciferase, Activity Assay, Microscopy, Staining, Labeling, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transfection, Incubation

Journal: Genes & Development

Article Title: Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5

doi: 10.1101/gad.252676.114

Figure Lengend Snippet: Dlg5 loss compromises Hh signal response without affecting the formation of the primary cilium or the ciliary accumulation of Smo. ( A ) Western blot (WB) analysis of Gli1 levels in Shh-stimulated Dlg5 +/− and Dlg5 −/− MEFs. Shh induces the accumulation of Gli1 in Dlg5 +/− cells, whereas Dlg5 −/− cells exhibit significantly reduced Gli1 protein levels in response to Shh. ( B ) Western blot analysis of Gli1 levels in Shh-stimulated Dlg5 −/− MEFs either uninfected or infected with LAP-Dlg5. Introduction of Dlg5 into Dlg5 −/− cells via retroviral transduction restores Gli1 protein accumulation in cells stimulated with Shh. ( C ) Dlg5 +/− and Dlg5 −/− MEFs were stimulated for 24 h with the indicated treatments, and Gli1 levels were analyzed by Western blot. Neither ShhN conditioned medium, 200 nM SAG1.5, nor a combination of 5 µM 20(S)/22(S)-hydroxycholesterol was capable of inducing Gli1 in Dlg5 −/− MEFs to levels comparable in Dlg5 +/− MEFs. ( D ) Western blot analysis of the effect of Dlg5 knockdown on Gli1 levels in unstimulated Ptch −/− and Sufu −/− MEFs. Depletion of Dlg5 in Ptch −/− MEFs resulted in a decrease in Gli1 protein, but a similar knockdown of Dlg5 in Sufu −/− MEFs had no appreciable effect on Gli1. ( E , F ) Microscopy analysis of Dlg5 +/− and Dlg5 −/− MEFs that were subjected to overnight serum starvation and subsequently stained with an antibody against acetylated tubulin (red) to label primary cilia. DAPI-labeled nuclei are outlined using white dashed circles, and primary cilia are indicated by asterisks. The fraction of nuclei associated with a primary cilium in each cell line is quantified in F . All error bars are SD. n > 95 nuclei. Data were analyzed using a two-tailed unpaired t -test ( P = 0.46). There was no significant difference in the frequency of ciliation in Dlg5 +/− and Dlg5 −/− cells. ( G , H ) Microscopy analysis of Shh-stimulated uninfected or LAP-Dlg5-infected Dlg5 −/− MEFs stained with antibodies against acetylated tubulin (red) and Smo (green). Images shown are shifted overlays. The fraction of primary cilia that overlaps with Smo is quantified and plotted in H . All error bars are SD. n > 43 cilia. Data were analyzed using a two-tailed unpaired t -test ( P = 0.36). The fraction of cilia that contained Smo in response to Shh was not significantly different in the two cell populations.

Article Snippet: SAG1.5 was purchased from Enzo Life Sciences, DDM and CHS were purchased from Affymetrix, and Cytochalasin B and nocodazole were purchased from Sigma.

Techniques: Western Blot, Infection, Transduction, Microscopy, Staining, Labeling, Two Tailed Test

Journal:

Article Title: Clostridium septicum Alpha-Toxin Is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

doi: 10.1128/IAI.70.8.4353-4361.2002

Figure Lengend Snippet: AT binds to GPI-anchored parasite proteins. A Triton X-114 extract of tachyzoites (T) was incubated with or without PI-PLC and phase partitioned into detergent (D) and aqueous (Aq) phases. The fractions were analyzed by SDS-PAGE under nonreducing (A) or reducing (B) conditions. The gels were either silver stained or transferred to nitrocellulose for toxin overlay or SAG1 Western blotting. Under nonreducing conditions, AT binds to proteins with approximate molecular masses of 54, 38, and 32 kDa and multiple lower-molecular-mass (∼18 to 25 kDa) proteins (arrowheads). All of these toxin-binding proteins (with the exception of the 54-kDa protein) partition into the detergent phase in the absence of PI-PLC treatment and into the aqueous phase after incubation with PI-PLC. AT binds significantly less protein after delipidation, a property that can be reversed by running the samples under reducing conditions. The major silver-stained protein in the detergent phase (asterisk) corresponds to SAG1. A SAG1 Western blot could not be run under reducing conditions, because the MAb does not recognize reduced protein. Numbers on the left indicate molecular mass in kilodaltons.

Article Snippet: The MAbs used were MAb G11-9 against SAG1 (Argene, Inc., Varihes, France), MAb 17.9 against GRA6 ( 10 ), and MAb 45.15 (K. L. Carey and G.E.W., unpublished data) against the inner membrane complex (IMC) protein IMC1 ( 30 ).

Techniques: Incubation, SDS Page, Staining, Western Blot, Binding Assay

Journal:

Article Title: Clostridium septicum Alpha-Toxin Is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

doi: 10.1128/IAI.70.8.4353-4361.2002

Figure Lengend Snippet: (A) AT and aerolysin bind to SAG1. Triton X-114 detergent-phase fractions from RH, P(LK), and P(LK)sag1 parasites were analyzed by SDS-PAGE followed by silver staining, AT overlay, aerolysin overlay, and immunoblotting with MAbs against SAG1 and SAG3. The SAG1 and SAG3 immunoblots were performed sequentially, and the data were combined digitally into the single panel shown. A 32-kDa toxin-binding band is observed in RH and P(LK) extracts that comigrates precisely with SAG1 on the corresponding immunoblot. Relatively more aerolysin binds to this protein [in both RH and P(LK) extracts] than does AT. SAG1-deficient parasites lack both immunoreactive SAG1 (as expected) and the 32-kDa toxin-binding protein. No other differences in the toxin-binding profiles were observed between P(LK) and the SAG1-deficient P(LK) parasites. Note that an additional 33-kDa AT-binding protein (asterisk) was observed in P(LK)sag1 parasites that was not observed in RH parasites. The P(LK) and P(LK)sag1 parasites also contained two aerolysin-binding bands not detected in RH parasites (arrowheads). Within each panel, equal amounts of protein were loaded per lane. Numbers on the left indicate molecular mass in kilodaltons. (B) AT and aerolysin bind to SAG3. Triton X-114 detergent-phase extracts from RH(ST) and SAG3-deficient RH(ST) parasites were analyzed as described above. A 38-kDa toxin-binding band is seen in RH(ST) extracts that comigrates precisely with SAG3 on the immunoblot. This 38-kDa toxin-binding band is absent in the RH(ST)ΔSAG3 parasites, which, as expected, also lack immunoreactive SAG3. Numbers on the left indicate molecular mass in kilodaltons.

Article Snippet: The MAbs used were MAb G11-9 against SAG1 (Argene, Inc., Varihes, France), MAb 17.9 against GRA6 ( 10 ), and MAb 45.15 (K. L. Carey and G.E.W., unpublished data) against the inner membrane complex (IMC) protein IMC1 ( 30 ).

Techniques: SDS Page, Silver Staining, Western Blot, Binding Assay

Journal:

Article Title: Clostridium septicum Alpha-Toxin Is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

doi: 10.1128/IAI.70.8.4353-4361.2002

Figure Lengend Snippet: Characterization of AT-treated tachyzoites by immunofluorescence and differential interference contrast (DIC) microscopy. Tachyzoites were incubated with 10 nM AT for 30, 60, or 240 min. Fixed and permeabilized parasites were then dual labeled with fluorescently conjugated MAbs against the GPI-anchored surface protein SAG1 and the inner membrane complex protein IMC1. After 30 min of AT treatment, a distinct vacuole was observed in most cells (arrowhead), and membrane protrusions were apparent. Between 60 and 240 min, massive membrane blebs appeared (arrows). The membrane blebs exhibited peripheral staining with the anti-SAG1 antibody, but not with the antibody against the IMC antigen; the distribution of the IMC antigen appeared unaffected by AT treatment. Scale bar, 5 μm.

Article Snippet: The MAbs used were MAb G11-9 against SAG1 (Argene, Inc., Varihes, France), MAb 17.9 against GRA6 ( 10 ), and MAb 45.15 (K. L. Carey and G.E.W., unpublished data) against the inner membrane complex (IMC) protein IMC1 ( 30 ).

Techniques: Immunofluorescence, Microscopy, Incubation, Labeling, Staining

Journal:

Article Title: Clostridium septicum Alpha-Toxin Is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

doi: 10.1128/IAI.70.8.4353-4361.2002

Figure Lengend Snippet: Immunofluorescence analysis of fixed and permeabilized versus nonpermeabilized AT-treated tachyzoites. Tachyzoites were treated for 240 min with 10 nM AT, fixed, and processed for immunofluorescence with (+TX-100) or without (−TX-100) detergent (Triton X-100) permeabilization. Parasites were dual labeled with MAbs against the surface protein SAG1 and the dense granule protein GRA6 (A) or SAG1 and IMC1 (B). Arrows indicate toxin-induced membrane blebs, and arrowheads point to sites at which plasma membrane had ruptured. Scale bar, 5 μm.

Article Snippet: The MAbs used were MAb G11-9 against SAG1 (Argene, Inc., Varihes, France), MAb 17.9 against GRA6 ( 10 ), and MAb 45.15 (K. L. Carey and G.E.W., unpublished data) against the inner membrane complex (IMC) protein IMC1 ( 30 ).

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants

doi: 10.1101/2024.10.15.618526

Figure Lengend Snippet: Immunofluorescence analysis of anti-FOS specificity using Toxoplasma infected HFFs. Monolayers of human foreskin fibroblasts were infected with parasites, fixed, and imaged using Super-Resolution Structured Illumination Microscopy (SR-SIM). (A) HFFs containing wild-type RHΔΔ or TgSPYΔ parasites were probed with affinity purified rabbit anti-FOS (1 µg/ml) and mouse anti-SAG1 followed by Alexa Fluor-488 goat anti-rabbit IgG and Alexa Fluor-594 goat anti-mouse IgG to localize O-Fuc and outline the parasites, respectively. DAPI (blue) was used to visualize parasite and host cell nuclei (an example is labeled). An instance of 2 parasites occupying a parasitophorous vacuole within an HFF is outlined with a white circle. In the lower row, RHΔΔ samples were probed with anti-FOS in the presence of 0.2 M αMeFuc. Maximum intensity projections are shown. (B) Same as panel A, except that 1 µg/ml AAL-biotin and Alexa Fluor-594 streptavidin were used in place of anti-FOS. Scale bars: 5 µm. See Fig. S1 for corresponding probing with anti-FOT. (C) Same as above, except that samples were probed with anti-FOS and either AAL-biotin or anti-PLP6 (1:5000), a marker of nuclear epichromatin. Representative single z-slices of the nuclear regions are shown. See Fig. S2 for additional images with anti-FOS/T. (D) Pearson’s correlation coefficients for colocalization probes in panel C. Each point represents a single cell (n>30), and means are represented with a horizontal bar. The difference was significant using an unpaired, two-tailed t-test (****p< 0.0001). Scale bars: 5 µm.

Article Snippet: HFF monolayers prepared on 12 mm diameter glass coverslips in 24-well plates were infected with tachyzoites and later fixed in cold methanol for 2 min, washed thrice with PBS (Corning 21-040-CV), and incubated in Blocking Buffer (5% (v/v) FBS, 5% (v/v) normal goat serum in PBS) for an additional 10 min. Coverslips were exposed to the following primary antibodies diluted in Blocking Buffer: affinity purified rabbit anti-FOS (1 µg/ml), rabbit anti-FOT (0.3 µg/ml), mouse anti-SAG1 (1:5000, MyBioSource), mouse anti-PLP6 (1:10,000, Millipore Sigma), and/or biotinylated Aleuria Aurantia Lectin (AAL) premixed with Alexa Flour 594 streptavidin (1:250, Vector Labs), for 1 h at 22°C.

Techniques: Immunofluorescence, Infection, Microscopy, Affinity Purification, Labeling, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: A phenotypic screen using splitCas9 identifies essential genes required for actin regulation during host cell egress and invasion by Toxoplasma gondii

doi: 10.1101/2021.09.24.461619

Figure Lengend Snippet: a, Schematic of the splitCas9-system. Transgenic parasites are generated co-expressing the two splitCas9 subunits together with a single-guide RNA (sgRNA). Upon addition of rapamycin the two subunits dimerise, leading to reconstituted Cas9 activity and therefore to disruption of the gene targeted by the sgRNA. b, Analysis of RHsCas9-gap40 parasites that were treated with 50 nM rapamycin for 48h before fixation using indicated antibodies. Scale bars are 5μm. Three distinct phenotypes can be observed: the gap40 phenotype, as described previously with collapsed IMC and loss of GAP40 expression (top panel, asterisk); a phenotype where some parasites within the PV are aberrant (bottom panel); and parasites with normal IMC and GAP40 localisation (top panel, arrow). c, Quantification of gap40 phenotypes shown in (a) in indicated parasite strains. Parasites were induced with or without rapamycin for the indicated time and fixed 48 h post infection. Only parasites expressing both the gap40sgRNA and the sCas9 system presented a gap40 phenotype after induction. Parasites were treated with rapamycin for 1h or the whole growth period of 48h as indicated. Data represents three independent experiments. For each condition 100 vacuoles were counted (total n=300). Average and standard deviation (SD) are represented. d, Analysis of RHsCas9-sag1 parasites that were treated with 50 nM rapamycin for 48h before fixation using indicated antibodies. Nuclei were stained with DAPI. Scale bars are 5μm. Three distinct phenotypes can be observed: healthy vacuoles lacking SAG1 (bottom panel, arrow); and parasites lacking SAG1 while displaying aberrant nuclear and cellular morphology (bottom panel, asterisk). e, Quantification of the phenotypes shown in (d). Aberrant nuclei and cellular morphology were observed only when sag1 was disrupted (KO) by sCas9 activation (1 st lytic cycle). Abundance of non-healthy parasites was reduced to background levels when induced RHsCas9- sag1 parasites were mechanically lysed, transferred onto fresh host cells and grown again for 48h in a second lytic cycle (2 nd generation; total incubation of 96h). Parasites were treated with rapamycin for 1h or for the whole growth period of 48h as indicated. Data represent three independent experiments. For each condition, 100 vacuoles were counted (total n=300). Average and standard deviation (SD) are represented. f, Analysis of indicator parasites expressing sgRNA targeting sag1. Disruption of sag1 has no effect on the F-actin network. Scale bars are 5 μm. g, Analysis of indicator parasites expressing indicated sgRNAs. IFA depicting the effect of drpA (RHsCas9- drpA ), or adf (RHsCas9- adf ), act1 (RHsCas9- act1) and frm2 (RHsCas9- frm2 ) disruption on actin network and apicoplast segregation. To achieve gene disruption (KO), parasites were incubated with 50 nM rapamycin for 1h and fixed after 48h.Nuclei were stained with DAPI. Scale bars are 5 μm. h, Depiction of indicator parasites co-expressing CbEm, FNR-RFP and the sCas9-subunits. Scale bar is 5 μm.

Article Snippet: The resulting plasmids were confirmed by sequencing. pU6- sag1 gRNA-scaffold and pU6-gap40 gRNA-scaffold sequence was synthesised and cloned into a backbone vector containing the DHFR resistance cassette by GeneScript.

Techniques: Transgenic Assay, Generated, Expressing, Activity Assay, Disruption, Infection, Standard Deviation, Staining, Activation Assay, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Application of Toxoplasma gondii -specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis

doi: 10.3389/fcimb.2022.1029768

Figure Lengend Snippet: The recombinant SAG1, GRA7 and BAG1 proteins of T. gondii was expressed. SDS-PAGE analysis showed the rSAG1, rGRA7, rBAG1 and GST proteins.

Article Snippet: Amino acid sequence alignment and phylogenetic analyses for Tg SAG1, Tg GRA7, and Tg BAG1 with the related cycle-forming organizations ( Neospora caninum , Besnoitia species , Sarcocystis species, etc.) were constructed using the maximum lifestyle statistical method and bootstrap analysis with 500 replications in MEGA7, and the sequences included T. gondii SAG1 (AFO54849.1), N. caninum SAG1 (AAD25091.1), Sarcocystis neurona SAG1 (AAK40366.1), T. gondii GRA7 (ABE69193.1), N. caninum GRA7 (AFB77190.1), Besnoitia besnoiti GRA7 (XP_029218567.1), T. gondii BAG1 (XP_002365116.1), N. caninum BAG1 (BAI44436.1), and B. besnoiti BAG1 (XP_029221932.1).

Techniques: Recombinant, SDS Page

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Application of Toxoplasma gondii -specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis

doi: 10.3389/fcimb.2022.1029768

Figure Lengend Snippet: Detection of T. gondii IgG (A, B) and IgM (C, D) antibodies in various animals in the Qinghai-Tibet Plateau by the indirect ELISA methods based on SAG1, GRA7 and BAG1 antigens in this study.

Article Snippet: Amino acid sequence alignment and phylogenetic analyses for Tg SAG1, Tg GRA7, and Tg BAG1 with the related cycle-forming organizations ( Neospora caninum , Besnoitia species , Sarcocystis species, etc.) were constructed using the maximum lifestyle statistical method and bootstrap analysis with 500 replications in MEGA7, and the sequences included T. gondii SAG1 (AFO54849.1), N. caninum SAG1 (AAD25091.1), Sarcocystis neurona SAG1 (AAK40366.1), T. gondii GRA7 (ABE69193.1), N. caninum GRA7 (AFB77190.1), Besnoitia besnoiti GRA7 (XP_029218567.1), T. gondii BAG1 (XP_002365116.1), N. caninum BAG1 (BAI44436.1), and B. besnoiti BAG1 (XP_029221932.1).

Techniques: Indirect ELISA

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Application of Toxoplasma gondii -specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis

doi: 10.3389/fcimb.2022.1029768

Figure Lengend Snippet: Seroprevalence of specific- T. gondii IgG and IgM antibodies in various animals.

Article Snippet: Amino acid sequence alignment and phylogenetic analyses for Tg SAG1, Tg GRA7, and Tg BAG1 with the related cycle-forming organizations ( Neospora caninum , Besnoitia species , Sarcocystis species, etc.) were constructed using the maximum lifestyle statistical method and bootstrap analysis with 500 replications in MEGA7, and the sequences included T. gondii SAG1 (AFO54849.1), N. caninum SAG1 (AAD25091.1), Sarcocystis neurona SAG1 (AAK40366.1), T. gondii GRA7 (ABE69193.1), N. caninum GRA7 (AFB77190.1), Besnoitia besnoiti GRA7 (XP_029218567.1), T. gondii BAG1 (XP_002365116.1), N. caninum BAG1 (BAI44436.1), and B. besnoiti BAG1 (XP_029221932.1).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Application of Toxoplasma gondii -specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis

doi: 10.3389/fcimb.2022.1029768

Figure Lengend Snippet: Specific- T. gondii IgG-positive animals based on among SAG1, GRA7 and BAG1 proteins.

Article Snippet: Amino acid sequence alignment and phylogenetic analyses for Tg SAG1, Tg GRA7, and Tg BAG1 with the related cycle-forming organizations ( Neospora caninum , Besnoitia species , Sarcocystis species, etc.) were constructed using the maximum lifestyle statistical method and bootstrap analysis with 500 replications in MEGA7, and the sequences included T. gondii SAG1 (AFO54849.1), N. caninum SAG1 (AAD25091.1), Sarcocystis neurona SAG1 (AAK40366.1), T. gondii GRA7 (ABE69193.1), N. caninum GRA7 (AFB77190.1), Besnoitia besnoiti GRA7 (XP_029218567.1), T. gondii BAG1 (XP_002365116.1), N. caninum BAG1 (BAI44436.1), and B. besnoiti BAG1 (XP_029221932.1).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Application of Toxoplasma gondii -specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis

doi: 10.3389/fcimb.2022.1029768

Figure Lengend Snippet: Specific- T. gondii IgM-positive animals based on among SAG1, GRA7 and BAG1 proteins.

Article Snippet: Amino acid sequence alignment and phylogenetic analyses for Tg SAG1, Tg GRA7, and Tg BAG1 with the related cycle-forming organizations ( Neospora caninum , Besnoitia species , Sarcocystis species, etc.) were constructed using the maximum lifestyle statistical method and bootstrap analysis with 500 replications in MEGA7, and the sequences included T. gondii SAG1 (AFO54849.1), N. caninum SAG1 (AAD25091.1), Sarcocystis neurona SAG1 (AAK40366.1), T. gondii GRA7 (ABE69193.1), N. caninum GRA7 (AFB77190.1), Besnoitia besnoiti GRA7 (XP_029218567.1), T. gondii BAG1 (XP_002365116.1), N. caninum BAG1 (BAI44436.1), and B. besnoiti BAG1 (XP_029221932.1).

Techniques: