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    Structured Review

    Thermo Fisher synthetic aβ preparations
    The β-amyloid-inducing activity is partly proteinase K-resistant Brain extracts from an aged APP23 tg mouse (Tg extract), an aged non-tg mouse (Wt extract) spiked with synthetic <t>Aβ</t> fibrils (syn. Aβ fibrils in Wt extract; 10µM Aβ), and synthetic Aβ fibrils in PBS (syn. Aβ fibrils in PBS; 10µM) were treated with 50µg/ml proteinase K (PK) for 0min, 30min, 1h, or 2h at 37°C. ( A ) Silver staining of a 12% Bis-Tris NuPage® gel for total protein reveals the digestion of the majority of proteins in the brain extracts and of synthetic Aβ fibrils already after 30 min PK treatment. ( B ) Immunoblot analysis with an antibody specific to human Aβ (6E10) shows that Aβ in the Tg extract is largely PK-resistant for up to 2h. Aβ in the spiked Wt extract revealed some, but significantly less PK-resistance, whereas synthetic Aβ fibrils in PBS were almost completely digested by the PK treatment. ( C ) Densitometric quantification of Aβ-immunoblots after PK treatment of 5 Tg extracts [each from a different animal] and 3 independent synthetic Aβ fibril preparations are shown. Each preparation was tested at least 3 times and the mean was taken. Aβ concentration at time point 0 was designated as 100%. Indicated is the mean ± SEM. Repeated Measures 2-way ANOVA revealed a significant difference between the groups (F (2,34) = 112.2). Multiple Bonferroni post hoc tests showed significances after PK digestion for all group comparisons, *** p
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    Images

    1) Product Images from "Soluble A? seeds are potent inducers of cerebral ?-amyloid deposition"

    Article Title: Soluble A? seeds are potent inducers of cerebral ?-amyloid deposition

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.3088-11.2011

    The β-amyloid-inducing activity is partly proteinase K-resistant Brain extracts from an aged APP23 tg mouse (Tg extract), an aged non-tg mouse (Wt extract) spiked with synthetic Aβ fibrils (syn. Aβ fibrils in Wt extract; 10µM Aβ), and synthetic Aβ fibrils in PBS (syn. Aβ fibrils in PBS; 10µM) were treated with 50µg/ml proteinase K (PK) for 0min, 30min, 1h, or 2h at 37°C. ( A ) Silver staining of a 12% Bis-Tris NuPage® gel for total protein reveals the digestion of the majority of proteins in the brain extracts and of synthetic Aβ fibrils already after 30 min PK treatment. ( B ) Immunoblot analysis with an antibody specific to human Aβ (6E10) shows that Aβ in the Tg extract is largely PK-resistant for up to 2h. Aβ in the spiked Wt extract revealed some, but significantly less PK-resistance, whereas synthetic Aβ fibrils in PBS were almost completely digested by the PK treatment. ( C ) Densitometric quantification of Aβ-immunoblots after PK treatment of 5 Tg extracts [each from a different animal] and 3 independent synthetic Aβ fibril preparations are shown. Each preparation was tested at least 3 times and the mean was taken. Aβ concentration at time point 0 was designated as 100%. Indicated is the mean ± SEM. Repeated Measures 2-way ANOVA revealed a significant difference between the groups (F (2,34) = 112.2). Multiple Bonferroni post hoc tests showed significances after PK digestion for all group comparisons, *** p
    Figure Legend Snippet: The β-amyloid-inducing activity is partly proteinase K-resistant Brain extracts from an aged APP23 tg mouse (Tg extract), an aged non-tg mouse (Wt extract) spiked with synthetic Aβ fibrils (syn. Aβ fibrils in Wt extract; 10µM Aβ), and synthetic Aβ fibrils in PBS (syn. Aβ fibrils in PBS; 10µM) were treated with 50µg/ml proteinase K (PK) for 0min, 30min, 1h, or 2h at 37°C. ( A ) Silver staining of a 12% Bis-Tris NuPage® gel for total protein reveals the digestion of the majority of proteins in the brain extracts and of synthetic Aβ fibrils already after 30 min PK treatment. ( B ) Immunoblot analysis with an antibody specific to human Aβ (6E10) shows that Aβ in the Tg extract is largely PK-resistant for up to 2h. Aβ in the spiked Wt extract revealed some, but significantly less PK-resistance, whereas synthetic Aβ fibrils in PBS were almost completely digested by the PK treatment. ( C ) Densitometric quantification of Aβ-immunoblots after PK treatment of 5 Tg extracts [each from a different animal] and 3 independent synthetic Aβ fibril preparations are shown. Each preparation was tested at least 3 times and the mean was taken. Aβ concentration at time point 0 was designated as 100%. Indicated is the mean ± SEM. Repeated Measures 2-way ANOVA revealed a significant difference between the groups (F (2,34) = 112.2). Multiple Bonferroni post hoc tests showed significances after PK digestion for all group comparisons, *** p

    Techniques Used: Activity Assay, Silver Staining, Western Blot, Concentration Assay

    Extended sonication increases β-amyloid-inducing activity Brain extracts (10% [w/v]) from aged APP23 mice (Tg extract) were subjected to additional sonication (3 × 20sec). ( A ) Immunoblot analysis with an antibody specific to human Aβ reveals no change in total Aβ between the original Tg extract (−) and the extract with extended sonication (+). However, more Aβ was found in the supernatant (SN) of the extra-sonicated extract after ultracentrifugation (100,000 × g; 30 min) (long exposure). ( B–C ) In vivo seeding activity of the original and extra-sonicated extracts was tested by intrahippocampal injections into young, pre-depositing APP23 mice. Brains were immunohistochemically analyzed for Aβ deposition 4 months post-injection. Shown is the dentate gyrus of the hippocampus. Injection of original Tg extract ( B ) induced robust congophilic Aβ deposition with a filamentous and dense pattern, while Aβ induction with the extra-sonicated Tg extract generated more punctate and small deposits ( C ). The insert shows double labeling of Aβ immunoreactivity and Congo red binding of the induced β-amyloid. Scale bars: 200µm and 20µm. ( D ) Stereological quantification of Aβ load in the hippocampus revealed a significant increase in β-amyloid induction by extended sonication. (n = 5–6 mice per group; mean ± SEM, t[9]=3.188; * p
    Figure Legend Snippet: Extended sonication increases β-amyloid-inducing activity Brain extracts (10% [w/v]) from aged APP23 mice (Tg extract) were subjected to additional sonication (3 × 20sec). ( A ) Immunoblot analysis with an antibody specific to human Aβ reveals no change in total Aβ between the original Tg extract (−) and the extract with extended sonication (+). However, more Aβ was found in the supernatant (SN) of the extra-sonicated extract after ultracentrifugation (100,000 × g; 30 min) (long exposure). ( B–C ) In vivo seeding activity of the original and extra-sonicated extracts was tested by intrahippocampal injections into young, pre-depositing APP23 mice. Brains were immunohistochemically analyzed for Aβ deposition 4 months post-injection. Shown is the dentate gyrus of the hippocampus. Injection of original Tg extract ( B ) induced robust congophilic Aβ deposition with a filamentous and dense pattern, while Aβ induction with the extra-sonicated Tg extract generated more punctate and small deposits ( C ). The insert shows double labeling of Aβ immunoreactivity and Congo red binding of the induced β-amyloid. Scale bars: 200µm and 20µm. ( D ) Stereological quantification of Aβ load in the hippocampus revealed a significant increase in β-amyloid induction by extended sonication. (n = 5–6 mice per group; mean ± SEM, t[9]=3.188; * p

    Techniques Used: Sonication, Activity Assay, Mouse Assay, In Vivo, Injection, Generated, Labeling, Binding Assay

    2) Product Images from "Targeted Ablation of Crb1 and Crb2 in Retinal Progenitor Cells Mimics Leber Congenital Amaurosis"

    Article Title: Targeted Ablation of Crb1 and Crb2 in Retinal Progenitor Cells Mimics Leber Congenital Amaurosis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003976

    Abnormal layering in Crb1Crb2 cKO retinas. Histological sections of P14 (A), 1M (B), 3M (C) and 6M (D) old control (left; Crb1 +/− Crb2 F/+ cKO), Crb1 +/− Crb2 cKO (middle) and Crb1Crb2 cKO (right). Crb1Crb2 cKO retinas had a thick ganglion cell layer and a second broad nuclear layer separated by the inner plexiform layer. Crb1 +/− Crb2 cKO had perturbed outer and inner nuclear layers. Ectopic localization of dark-pigmented photoreceptors (white arrows), ganglion/inner nuclear layer cells (white asterisks) and rosettes of photoreceptors (black arrowheads) was visible in the two mutant retinas ( Figure S5B ,D). Both mutant retinas degenerated rapidly with age. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; (E,F) Electron microscopic pictures of 1M old Crb1Crb2 cKO retinas. Some complete segments (E, white asterix), adherens junctions (E, black arrow) and centrioles of cilium (E, bracket) or ribbon synapses with vesicles on the two sides of the cleft (F, black asterix) were identified but in ectopic locations. (G) The thickness of 4–5 control and Crb1Crb2 cKO retinas from P8 to P360. Crb1Crb2 cKO retinas had a thicker retina than littermate controls at P10 and P14, followed by progressive thinning and degeneration. (H) The cleaved caspase 3 positive apoptotic cells were counted at P10, P14 and 3M from 20–30 sections of 3 littermate controls and Crb1Crb2 cKO whole retinas. Mutant retinas showed an increase in the number of apoptotic cells. Data are presented as mean ± s.e.m. *P
    Figure Legend Snippet: Abnormal layering in Crb1Crb2 cKO retinas. Histological sections of P14 (A), 1M (B), 3M (C) and 6M (D) old control (left; Crb1 +/− Crb2 F/+ cKO), Crb1 +/− Crb2 cKO (middle) and Crb1Crb2 cKO (right). Crb1Crb2 cKO retinas had a thick ganglion cell layer and a second broad nuclear layer separated by the inner plexiform layer. Crb1 +/− Crb2 cKO had perturbed outer and inner nuclear layers. Ectopic localization of dark-pigmented photoreceptors (white arrows), ganglion/inner nuclear layer cells (white asterisks) and rosettes of photoreceptors (black arrowheads) was visible in the two mutant retinas ( Figure S5B ,D). Both mutant retinas degenerated rapidly with age. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; (E,F) Electron microscopic pictures of 1M old Crb1Crb2 cKO retinas. Some complete segments (E, white asterix), adherens junctions (E, black arrow) and centrioles of cilium (E, bracket) or ribbon synapses with vesicles on the two sides of the cleft (F, black asterix) were identified but in ectopic locations. (G) The thickness of 4–5 control and Crb1Crb2 cKO retinas from P8 to P360. Crb1Crb2 cKO retinas had a thicker retina than littermate controls at P10 and P14, followed by progressive thinning and degeneration. (H) The cleaved caspase 3 positive apoptotic cells were counted at P10, P14 and 3M from 20–30 sections of 3 littermate controls and Crb1Crb2 cKO whole retinas. Mutant retinas showed an increase in the number of apoptotic cells. Data are presented as mean ± s.e.m. *P

    Techniques Used: Mutagenesis

    CRB1 and CRB2 acts on the proliferative signalling pathways. Transcript levels measured by quantitative PCR at E17.5 (A) and P3 (C) in 3–6 control and Crb1Crb2 cKO retinas showed changes in Notch1, YAP, sonic hedgehogs and cell cycle genes at E17,5 whereas at P1 these genes were not significantly changed except Hey1 and Smoothened. Quantification of protein levels of control and Crb1Crb2 cKO retinal lysates (N = 3–5 for each Western blot and Western blots were repeated 2–4 times) at E17.5 (B) and P1 (D). Protein levels of YAP and pYAP were reduced at E17.5 and P1 whereas P120-catenin was increased and β-catenin and Kaiso unchanged at E17.5. Data are presented as mean ± s.e.m *P
    Figure Legend Snippet: CRB1 and CRB2 acts on the proliferative signalling pathways. Transcript levels measured by quantitative PCR at E17.5 (A) and P3 (C) in 3–6 control and Crb1Crb2 cKO retinas showed changes in Notch1, YAP, sonic hedgehogs and cell cycle genes at E17,5 whereas at P1 these genes were not significantly changed except Hey1 and Smoothened. Quantification of protein levels of control and Crb1Crb2 cKO retinal lysates (N = 3–5 for each Western blot and Western blots were repeated 2–4 times) at E17.5 (B) and P1 (D). Protein levels of YAP and pYAP were reduced at E17.5 and P1 whereas P120-catenin was increased and β-catenin and Kaiso unchanged at E17.5. Data are presented as mean ± s.e.m *P

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot

    The number of late born cell types is increased in Crb1Crb2 cKO retinas. The distribution of early (A–C) and late born (D–F) cell types in the three layers was quantified as a percentage of each cell type in outer, inner and ganglion cell nuclear layer in the control (white bars), and top and bottom half of nuclear layer (tNL and bNL) and ganglion cell layer in the Crb1Crb2 cKO (black bars) retinas at P14 (3–4 different animals/genotype). The distribution of early-born ganglion cells (Brn3b), cone photoreceptors (cone arrestin) and cholinergic amacrine cells (choline acetyltransferase, ChAT) was slightly affected in contrast to late-born rod photoreceptors (rhodopsin), Müller cells (Sox9) and bipolar cells (Chx10), which were to a larger extent wrongly distributed in the two nuclear layers. (G,H) The number of cells for each cell types was quantified at P14 (G) and the rods at P10 (H) in 3–4 retinas of control and Crb1Crb2 cKO, and represented by the mean ± s.e.m. The number of early born cells was not affected whereas the number of late born cells was increased in Crb1Crb2 cKO compared to control retinas at P14 and the rods at P10. bNL, bottom nuclear layer; Calb, calbindin positive horizontal cells; CAR, cone arrestin; GCL, ganglion cell layer; GlyT1, glycinergic amacrine cells; INL, inner nuclear layer; IPL, Inner plexiform layer; ONL, outer nuclear layer; RHO, Rhodopsin; tNL, top nuclear layer. **P
    Figure Legend Snippet: The number of late born cell types is increased in Crb1Crb2 cKO retinas. The distribution of early (A–C) and late born (D–F) cell types in the three layers was quantified as a percentage of each cell type in outer, inner and ganglion cell nuclear layer in the control (white bars), and top and bottom half of nuclear layer (tNL and bNL) and ganglion cell layer in the Crb1Crb2 cKO (black bars) retinas at P14 (3–4 different animals/genotype). The distribution of early-born ganglion cells (Brn3b), cone photoreceptors (cone arrestin) and cholinergic amacrine cells (choline acetyltransferase, ChAT) was slightly affected in contrast to late-born rod photoreceptors (rhodopsin), Müller cells (Sox9) and bipolar cells (Chx10), which were to a larger extent wrongly distributed in the two nuclear layers. (G,H) The number of cells for each cell types was quantified at P14 (G) and the rods at P10 (H) in 3–4 retinas of control and Crb1Crb2 cKO, and represented by the mean ± s.e.m. The number of early born cells was not affected whereas the number of late born cells was increased in Crb1Crb2 cKO compared to control retinas at P14 and the rods at P10. bNL, bottom nuclear layer; Calb, calbindin positive horizontal cells; CAR, cone arrestin; GCL, ganglion cell layer; GlyT1, glycinergic amacrine cells; INL, inner nuclear layer; IPL, Inner plexiform layer; ONL, outer nuclear layer; RHO, Rhodopsin; tNL, top nuclear layer. **P

    Techniques Used:

    Retinal development is impaired in Crb1Crb2 cKO. (A–E) Histological sections from E13.5 to P5 control (left), Crb1 +/− Crb2 cKO (middle) and Crb1Crb2 cKO (right). From E13.5 onwards, disruption of the outer limiting membrane (A right, black arrowhead) accompanied with ectopic localization of cells extended in Crb1Crb2 cKO developing retinas (A,B right). At E17.5 and P1, in contrast to control retinas no proper ganglion cell layer was formed (C,D). The separation of the outer nuclear/photoreceptor layer formed around P5, which never happened in the Crb1Crb2 cKO retinas (E). Crb1 +/− Crb2 cKO retinas showed the first disruption in the outer limiting membrane at the periphery at E15.5 (B middle, black arrowhead), which progressively extended to the centre accompanied with rosette formation (B–E middle). Electron microscopic pictures from E17.5 littermate control (F) and Crb1Crb2 cKO (G) retinas. Control retinas showed an organized outer limiting membrane with adherens junctions (white arrowheads), retinal pigment epithelium and retinal nuclei alignments. Crb1Crb2 cKO retinas showed absence of layer organization and adherens junctions. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblast layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Scale bar, 100 µm (A–E); 5 µm (F,G).
    Figure Legend Snippet: Retinal development is impaired in Crb1Crb2 cKO. (A–E) Histological sections from E13.5 to P5 control (left), Crb1 +/− Crb2 cKO (middle) and Crb1Crb2 cKO (right). From E13.5 onwards, disruption of the outer limiting membrane (A right, black arrowhead) accompanied with ectopic localization of cells extended in Crb1Crb2 cKO developing retinas (A,B right). At E17.5 and P1, in contrast to control retinas no proper ganglion cell layer was formed (C,D). The separation of the outer nuclear/photoreceptor layer formed around P5, which never happened in the Crb1Crb2 cKO retinas (E). Crb1 +/− Crb2 cKO retinas showed the first disruption in the outer limiting membrane at the periphery at E15.5 (B middle, black arrowhead), which progressively extended to the centre accompanied with rosette formation (B–E middle). Electron microscopic pictures from E17.5 littermate control (F) and Crb1Crb2 cKO (G) retinas. Control retinas showed an organized outer limiting membrane with adherens junctions (white arrowheads), retinal pigment epithelium and retinal nuclei alignments. Crb1Crb2 cKO retinas showed absence of layer organization and adherens junctions. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblast layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Scale bar, 100 µm (A–E); 5 µm (F,G).

    Techniques Used:

    Retinal function in Crb1Crb2 mutant retinas is severely impaired. Retinal function in Crb1 KO (black), Crb2 cKO (green), Crb1 +/− Crb2 cKO (purple) and Crb1Crb2 cKO affected mice (red) based on single-flash electroretinogram data from 1M (A), and 3M (B) old animals. (left) Representative single-flash electroretinogram traces recorded from the indicated genotypes under scotopic (top) and photopic (bottom) conditions. (right) Scotopic (top) and photopic (bottom) b-wave amplitude data plotted as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quantile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. In Crb1 +/− Crb2 cKO and Crb1Crb2 cKO mice, the b-wave amplitude was already considerably reduced at 1M under both scotopic and photopic conditions, and declined even at 3M compared to Crb1 KO and Crb2 cKO.
    Figure Legend Snippet: Retinal function in Crb1Crb2 mutant retinas is severely impaired. Retinal function in Crb1 KO (black), Crb2 cKO (green), Crb1 +/− Crb2 cKO (purple) and Crb1Crb2 cKO affected mice (red) based on single-flash electroretinogram data from 1M (A), and 3M (B) old animals. (left) Representative single-flash electroretinogram traces recorded from the indicated genotypes under scotopic (top) and photopic (bottom) conditions. (right) Scotopic (top) and photopic (bottom) b-wave amplitude data plotted as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quantile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. In Crb1 +/− Crb2 cKO and Crb1Crb2 cKO mice, the b-wave amplitude was already considerably reduced at 1M under both scotopic and photopic conditions, and declined even at 3M compared to Crb1 KO and Crb2 cKO.

    Techniques Used: Mutagenesis, Mouse Assay

    Loss of CRB1 and CRB2 leads to cell cycle defects, increased proliferation and apoptosis. The number of mitotic cells immunostained with anti-phospho-Histone H3 (pH3; A) and apoptotic cells immunostained with cleaved caspase 3 (B) were quantified from E13.5 to P5. Crb1Crb2 cKO retinas showed a significant increased number of mitotic and apoptotic cells from E15.5 and E17.5 respectively in comparison to control retinas. At E17.5, the number of mitotic cells in the cell cycle using pH3 (M-phase), 30 min-pulse BrdU labelling (S-phase) and Ki67 immunostaining (all phases) was increased in Crb1Crb2 cKO retinas (C). Quantification at E17.5 showed that the number of early-born (Islet1, amacrine and ganglion cells) progenitor cells was not affected whereas late-born (Otx2, photoreceptors) progenitor cells was increased in Crb1Crb2 cKO compared to control retinas (D). Cell cycle exit index (F) was determined as the ratio of BrdU + /Ki67 − cells (no longer dividing) to total (24 hours) BrdU + cells. In Crb1Crb2 cKO retinas less cells exit the cell cycle in the BrdU labelled population. Data from 20 representative sections/pictures of whole retinas from 3–5 control and Crb1Crb2 cKO retinas are presented as mean ± s.e.m. Flow cytometry analysis of cell cycle in Crb1Crb2 cKO and control retinas at E17.5 (E), P1 (G) and P5 (H) revealed that only at E17.5 the proportion of cells in the different cell cycle phases is changed compared to control. *P
    Figure Legend Snippet: Loss of CRB1 and CRB2 leads to cell cycle defects, increased proliferation and apoptosis. The number of mitotic cells immunostained with anti-phospho-Histone H3 (pH3; A) and apoptotic cells immunostained with cleaved caspase 3 (B) were quantified from E13.5 to P5. Crb1Crb2 cKO retinas showed a significant increased number of mitotic and apoptotic cells from E15.5 and E17.5 respectively in comparison to control retinas. At E17.5, the number of mitotic cells in the cell cycle using pH3 (M-phase), 30 min-pulse BrdU labelling (S-phase) and Ki67 immunostaining (all phases) was increased in Crb1Crb2 cKO retinas (C). Quantification at E17.5 showed that the number of early-born (Islet1, amacrine and ganglion cells) progenitor cells was not affected whereas late-born (Otx2, photoreceptors) progenitor cells was increased in Crb1Crb2 cKO compared to control retinas (D). Cell cycle exit index (F) was determined as the ratio of BrdU + /Ki67 − cells (no longer dividing) to total (24 hours) BrdU + cells. In Crb1Crb2 cKO retinas less cells exit the cell cycle in the BrdU labelled population. Data from 20 representative sections/pictures of whole retinas from 3–5 control and Crb1Crb2 cKO retinas are presented as mean ± s.e.m. Flow cytometry analysis of cell cycle in Crb1Crb2 cKO and control retinas at E17.5 (E), P1 (G) and P5 (H) revealed that only at E17.5 the proportion of cells in the different cell cycle phases is changed compared to control. *P

    Techniques Used: Immunostaining, Flow Cytometry, Cytometry

    In vivo retinal imaging in Crb1Crb2 cKO mice. 12M old control Crb1 +/− Crb2 F/+ cKO (A–C) and 1M (D–F), 3M (G–I) and 6M (J–L) old Crb1Crb2 cKO mice were subjected to scanning laser ophthalmoscopy (A,D,G,J) and vertical spectral domain optical coherence tomography (B, E, H, K). C, F, I and L are magnifications of the boxes in B, E, H, and K respectively. At 1M, a disorganized retina with an abnormally thick ganglion cell layer, one plexiform and one nuclear layer was observed (E, asterisk in F). At 3M (G–I) and 6M (J–L), an obvious loss in the retinal thickness was ascertained as well as several fundus alterations (G,J). Abbreviations: AF, autofluorescence; d, dorsal; FA, Fluorescein angiography; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment/outer segment border; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; RF, Red-free; v, ventral.
    Figure Legend Snippet: In vivo retinal imaging in Crb1Crb2 cKO mice. 12M old control Crb1 +/− Crb2 F/+ cKO (A–C) and 1M (D–F), 3M (G–I) and 6M (J–L) old Crb1Crb2 cKO mice were subjected to scanning laser ophthalmoscopy (A,D,G,J) and vertical spectral domain optical coherence tomography (B, E, H, K). C, F, I and L are magnifications of the boxes in B, E, H, and K respectively. At 1M, a disorganized retina with an abnormally thick ganglion cell layer, one plexiform and one nuclear layer was observed (E, asterisk in F). At 3M (G–I) and 6M (J–L), an obvious loss in the retinal thickness was ascertained as well as several fundus alterations (G,J). Abbreviations: AF, autofluorescence; d, dorsal; FA, Fluorescein angiography; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment/outer segment border; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; RF, Red-free; v, ventral.

    Techniques Used: In Vivo, Imaging, Mouse Assay

    3) Product Images from "Coupling of 5S RNP rotation with maturation of functional centers during large ribosomal subunit assembly"

    Article Title: Coupling of 5S RNP rotation with maturation of functional centers during large ribosomal subunit assembly

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17534-5

    The CTD of Rpf2 is necessary as Nog2 particles transition to Rix1/Rea1 particles. a The 25S rRNA domain V and the Rpf2 subcomplex are in a flexible state and thus invisible (represented as the group in light colors) prior to rearrangements within the State E particle, including exit of labeled AFs from state E particles. b Stable anchoring of H74-H79, conformational changes in H68-70, as well as entry of Nog2, Rsa4, and Nop53, upon formation of Nog2 particles. c The Rix1/Rea1 particle is formed upon exit of the Rpf2-Rrs1 dimer, and entry of Sda1, the Rix1 subcomplex, and Rea1, as well as 5S RNP rotation. d In the absence of the CTD of Rpf2, the transition between the Nog2 and Rix1/Rea1 particles does not occur. For simplicity and clarity, only relevant AFs are shown and colored, and all r-proteins except rpL5 and rpL11 are omitted. Only partial densities for Sda1, Rea1, and the Rix1 subcomplex are available.
    Figure Legend Snippet: The CTD of Rpf2 is necessary as Nog2 particles transition to Rix1/Rea1 particles. a The 25S rRNA domain V and the Rpf2 subcomplex are in a flexible state and thus invisible (represented as the group in light colors) prior to rearrangements within the State E particle, including exit of labeled AFs from state E particles. b Stable anchoring of H74-H79, conformational changes in H68-70, as well as entry of Nog2, Rsa4, and Nop53, upon formation of Nog2 particles. c The Rix1/Rea1 particle is formed upon exit of the Rpf2-Rrs1 dimer, and entry of Sda1, the Rix1 subcomplex, and Rea1, as well as 5S RNP rotation. d In the absence of the CTD of Rpf2, the transition between the Nog2 and Rix1/Rea1 particles does not occur. For simplicity and clarity, only relevant AFs are shown and colored, and all r-proteins except rpL5 and rpL11 are omitted. Only partial densities for Sda1, Rea1, and the Rix1 subcomplex are available.

    Techniques Used: Labeling

    The conformation of Nog2 is altered in the C4 class of rpf2Δ255-344 particles. a The NTD of Nog2 is docked in the groove of H75 near H68 in wild-type particles and may affect the conformations of these helices, or otherwise be affected by them. b Atomic models for Nog2 in wild-type and C4 particles were aligned. The NTD of Nog2 is flexible and invisible in the C4 class of the rpf2Δ255-344 mutant particles. Conformational changes are visible in the GTPase domain of Nog2, where portions of the structure are deflected up to 4 Å.
    Figure Legend Snippet: The conformation of Nog2 is altered in the C4 class of rpf2Δ255-344 particles. a The NTD of Nog2 is docked in the groove of H75 near H68 in wild-type particles and may affect the conformations of these helices, or otherwise be affected by them. b Atomic models for Nog2 in wild-type and C4 particles were aligned. The NTD of Nog2 is flexible and invisible in the C4 class of the rpf2Δ255-344 mutant particles. Conformational changes are visible in the GTPase domain of Nog2, where portions of the structure are deflected up to 4 Å.

    Techniques Used: Mutagenesis

    The NTD of Rsa4 is invisible, and likely flexible in the C4 class of rpf2Δ255-344 . a The subunit interface view of density maps from the wild-type RPF2 and the C4 class of rpf2Δ255-344 mutant particles. Densities for Rsa4 were colored using Color Zone in Chimera. b Atomic models for Rsa4 from the wild-type and C4 class of the rpf2Δ255-344 mutant were fitted into density maps of the wild-type and the C4 class mutant. The first 34 amino acids of the NTD of the Rsa4 are flexible and invisible in the absence of the CTD of Rpf2. c Atomic models for Rsa4 and Nog2 from the wild-type and the C4 class are colored and labeled. For clarity, only relevant side chains are shown. Conformational changes in Nog2 may affect Rsa4, because Nog2 and Rsa4 are in close contact at two points (rectangles): at the NTD and the globular domain of Rsa4. Enlarged rectangles show contact points between Rsa4 and Nog2.
    Figure Legend Snippet: The NTD of Rsa4 is invisible, and likely flexible in the C4 class of rpf2Δ255-344 . a The subunit interface view of density maps from the wild-type RPF2 and the C4 class of rpf2Δ255-344 mutant particles. Densities for Rsa4 were colored using Color Zone in Chimera. b Atomic models for Rsa4 from the wild-type and C4 class of the rpf2Δ255-344 mutant were fitted into density maps of the wild-type and the C4 class mutant. The first 34 amino acids of the NTD of the Rsa4 are flexible and invisible in the absence of the CTD of Rpf2. c Atomic models for Rsa4 and Nog2 from the wild-type and the C4 class are colored and labeled. For clarity, only relevant side chains are shown. Conformational changes in Nog2 may affect Rsa4, because Nog2 and Rsa4 are in close contact at two points (rectangles): at the NTD and the globular domain of Rsa4. Enlarged rectangles show contact points between Rsa4 and Nog2.

    Techniques Used: Mutagenesis, Labeling

    AFs necessary for 5S rotation and export are affected in the rpf2Δ255-344 mutant. a SDS-PAGE of proteins in assembling 60S subunits from wild-type cells or from the rpf2Δ255-344 mutant shifted to 16 °C for 5 h. Pre-ribosomal particles were purified using AF Nog2 as a bait and protein constituents were separated by electrophoresis and stained with silver. Molecular weight standards, the Nog2 bait protein Nog2-Cbp (calmodulin-binding peptide left behind after TEV cleavage), and relevant AFs are labeled. b Samples from a were subjected to western blotting using antibodies against specific proteins. All samples were derived from the same experiment and western blottings were processed in parallel. c Samples prepared as described in a were used for iTRAQ (semi-quantitative mass spectrometry) to quantify relative changes in levels of AFs in the genomic rpf2Δ255-344 mutant compared with the wild-type strain. The ratios were normalized to levels of Nog2 (bait). The fold change is shown using bar graphs (orange) in log2 scale as an average of two biologically independent samples ( n = 2) for the genomic rpf2Δ255-344 mutant compared with the wild-type grown at 16 °C. Dot blots represent values for each biological replicate (red and blue). Source data are provided as a Source Data file.
    Figure Legend Snippet: AFs necessary for 5S rotation and export are affected in the rpf2Δ255-344 mutant. a SDS-PAGE of proteins in assembling 60S subunits from wild-type cells or from the rpf2Δ255-344 mutant shifted to 16 °C for 5 h. Pre-ribosomal particles were purified using AF Nog2 as a bait and protein constituents were separated by electrophoresis and stained with silver. Molecular weight standards, the Nog2 bait protein Nog2-Cbp (calmodulin-binding peptide left behind after TEV cleavage), and relevant AFs are labeled. b Samples from a were subjected to western blotting using antibodies against specific proteins. All samples were derived from the same experiment and western blottings were processed in parallel. c Samples prepared as described in a were used for iTRAQ (semi-quantitative mass spectrometry) to quantify relative changes in levels of AFs in the genomic rpf2Δ255-344 mutant compared with the wild-type strain. The ratios were normalized to levels of Nog2 (bait). The fold change is shown using bar graphs (orange) in log2 scale as an average of two biologically independent samples ( n = 2) for the genomic rpf2Δ255-344 mutant compared with the wild-type grown at 16 °C. Dot blots represent values for each biological replicate (red and blue). Source data are provided as a Source Data file.

    Techniques Used: Mutagenesis, SDS Page, Purification, Electrophoresis, Staining, Molecular Weight, Binding Assay, Labeling, Western Blot, Derivative Assay, Mass Spectrometry

    Changes in H68-70 are not caused by the absence of Sda1, which binds to H68-69. a Maturation of H68-H71 in wild-type cells during the transition from Nog2 particles to Rix1/Rea1 particles, then Nmd3 particles, and finally to mature 60S subunits (PDB IDs 3JCT [ https://www.wwpdb.org/pdb?id=pdb_00003jct ], 5FL8 [10.2210/pdb5FL8/pdb], 5APN [10.2210/pdb5APN/pdb], and 4V88 [10.2210/pdb4v88/pdb]). b Left: partial structure of Sda1 from the Rix1/Rea1 (Nog2 state 2) particle superimposed onto the structure of the Nog2 particle (PDB ID 3JCT [ https://www.wwpdb.org/pdb?id=pdb_00003jct ]). Sda1 is in close contact with H68 and H69 of the Nog2 particle. Right: Sda1 is in close contact with H68 and H69 in the Rix1/Rea1 particle (PBD ID 5FL8 [10.2210/pdb5FL8/pdb]), and the conformation of H69 is different from that in the Nog2 particle, presumably due to binding of Sda1. c Subunit interface view of density maps from the wild-type and E2 class of the Sda1-depleted particles. Densities for Rpf2, Rrs1 and H68-70 were colored using Color Zone in Chimera. No densities for H68-70 are missing in the E2 class compared with the wild-type Nog2 densities. d The atomic model for the wild-type particle was fitted into density maps of wild-type and E2 particles, respectively. Densities for Rpf2 and H68-70 are present in particles missing Sda1.
    Figure Legend Snippet: Changes in H68-70 are not caused by the absence of Sda1, which binds to H68-69. a Maturation of H68-H71 in wild-type cells during the transition from Nog2 particles to Rix1/Rea1 particles, then Nmd3 particles, and finally to mature 60S subunits (PDB IDs 3JCT [ https://www.wwpdb.org/pdb?id=pdb_00003jct ], 5FL8 [10.2210/pdb5FL8/pdb], 5APN [10.2210/pdb5APN/pdb], and 4V88 [10.2210/pdb4v88/pdb]). b Left: partial structure of Sda1 from the Rix1/Rea1 (Nog2 state 2) particle superimposed onto the structure of the Nog2 particle (PDB ID 3JCT [ https://www.wwpdb.org/pdb?id=pdb_00003jct ]). Sda1 is in close contact with H68 and H69 of the Nog2 particle. Right: Sda1 is in close contact with H68 and H69 in the Rix1/Rea1 particle (PBD ID 5FL8 [10.2210/pdb5FL8/pdb]), and the conformation of H69 is different from that in the Nog2 particle, presumably due to binding of Sda1. c Subunit interface view of density maps from the wild-type and E2 class of the Sda1-depleted particles. Densities for Rpf2, Rrs1 and H68-70 were colored using Color Zone in Chimera. No densities for H68-70 are missing in the E2 class compared with the wild-type Nog2 densities. d The atomic model for the wild-type particle was fitted into density maps of wild-type and E2 particles, respectively. Densities for Rpf2 and H68-70 are present in particles missing Sda1.

    Techniques Used: Binding Assay

    4) Product Images from "An Inducible Chaperone Adapts Proteasome Assembly to Stress"

    Article Title: An Inducible Chaperone Adapts Proteasome Assembly to Stress

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2014.06.017

    Adc17 Binds to Rpt6 and Assists Pairing of Rpt6 and Rpt3 Amino Termini (A) Yeast carrying the indicated plasmid combinations were spotted on selective medium lacking uracil, leucine, and histidine (−ULH) for yeast two-hybrid selection based on His3-auxotrophy. Growth on medium lacking uracil and leucine (−UL) served as a control. GAD, activation domain vector; GBD, DNA binding domain vector. (B) Left: schematics of Rpt6 domains. CC, coiled-coil. Right: Same as in (A) with indicated constructs. Growth on medium lacking uracil, leucine, histidine, and adenine (−ULHA) serves as stringent yeast two-hybrid selection. (C) Adc17 binds to Rpt6-NT. His-Adc17 and GST-Rpt6-NT (1–152) expressed in E. coli alone or together and purified on Ni-NTA (Ni) affinity resin or glutathione Sepharose 4B (GSH), resolved by SDS-PAGE, and stained with Coomassie blue. (D) Adc17 does not bind to Rpt3-NT. His-Adc17 and GST-Rpt3-NT (1–162) expressed in E. coli alone or together were purified on Ni-NTA affinity resin (Ni) or glutathione Sepharose 4B (GSH), resolved by SDS-PAGE, and stained with Coomassie blue. (E) Protein alignment of Adc17 from indicated species. The line indicates a predicted coiled-coil and the asterisk indicates a conserved leucine in the middle of the predicted coiled-coil. Identical or conserved residues are boxed in black and similar residues are in gray. (F) Yeast cells ( cim3-1) transformed with the indicated plasmids or empty vector (p416 GPD) were serially diluted, spotted on selective medium, and grown at indicated temperature. (G) Same as in (C) except that a point mutant of Adc17 (L93D) was used. (H) Rpt6-NT-MBP (1–140), Rpt3-NT-GST (1–156), and His-Adc17 were coexpressed in E. coli , purified on amylose resin, eluted, and analyzed by gel filtration. Left: chromatogram. Right: Coomassie-stained gels showing proteins analyzed from peak fractions indicated with “A” and “B.” The identity of the proteins was confirmed by mass spectrometry. ∗ , MBP. Representative results of at least three independent experiments are shown in each panel.
    Figure Legend Snippet: Adc17 Binds to Rpt6 and Assists Pairing of Rpt6 and Rpt3 Amino Termini (A) Yeast carrying the indicated plasmid combinations were spotted on selective medium lacking uracil, leucine, and histidine (−ULH) for yeast two-hybrid selection based on His3-auxotrophy. Growth on medium lacking uracil and leucine (−UL) served as a control. GAD, activation domain vector; GBD, DNA binding domain vector. (B) Left: schematics of Rpt6 domains. CC, coiled-coil. Right: Same as in (A) with indicated constructs. Growth on medium lacking uracil, leucine, histidine, and adenine (−ULHA) serves as stringent yeast two-hybrid selection. (C) Adc17 binds to Rpt6-NT. His-Adc17 and GST-Rpt6-NT (1–152) expressed in E. coli alone or together and purified on Ni-NTA (Ni) affinity resin or glutathione Sepharose 4B (GSH), resolved by SDS-PAGE, and stained with Coomassie blue. (D) Adc17 does not bind to Rpt3-NT. His-Adc17 and GST-Rpt3-NT (1–162) expressed in E. coli alone or together were purified on Ni-NTA affinity resin (Ni) or glutathione Sepharose 4B (GSH), resolved by SDS-PAGE, and stained with Coomassie blue. (E) Protein alignment of Adc17 from indicated species. The line indicates a predicted coiled-coil and the asterisk indicates a conserved leucine in the middle of the predicted coiled-coil. Identical or conserved residues are boxed in black and similar residues are in gray. (F) Yeast cells ( cim3-1) transformed with the indicated plasmids or empty vector (p416 GPD) were serially diluted, spotted on selective medium, and grown at indicated temperature. (G) Same as in (C) except that a point mutant of Adc17 (L93D) was used. (H) Rpt6-NT-MBP (1–140), Rpt3-NT-GST (1–156), and His-Adc17 were coexpressed in E. coli , purified on amylose resin, eluted, and analyzed by gel filtration. Left: chromatogram. Right: Coomassie-stained gels showing proteins analyzed from peak fractions indicated with “A” and “B.” The identity of the proteins was confirmed by mass spectrometry. ∗ , MBP. Representative results of at least three independent experiments are shown in each panel.

    Techniques Used: Plasmid Preparation, Selection, Activation Assay, Binding Assay, Construct, Purification, SDS Page, Staining, Transformation Assay, Mutagenesis, Filtration, Mass Spectrometry

    Adc17 Is Important for Proteasome Integrity (A) Native-PAGE (4.2%) of yeast extracts of wild-type (WT) or cim3-1 cultures, grown at 30°C or 37°C, revealed with the fluorogenic substrate Suc-LLVY-AMC. RP 2 CP, doubly capped proteasome; RPCP, singly capped proteasome. The alternative proteasome complex composed of the activator Bml10 bound to CP is indicated. Addition of 0.1% SDS activated the otherwise latent free CP. (B) Same as in (A) with cim3-1 cells transformed with the indicated plasmids or empty vector. RPT6 -complemented cells serve as a reference. (C) Native-PAGE of yeast extracts of cells of the indicated genotype, grown at 30°C. (D–F) Quantification of Rpt6 levels, normalized to control (either wild-type or Rpt6 complemented cells), are shown below Rpt6 blots. (D) Immunoblots of total extracts of wild-type or cim3-1 cells grown at 30°C or 37°C. (E) Same as in (D) with cim3-1 transformed with the indicated plasmids or empty vector. (F) Immunoblots of total extracts of cells of the indicated genotype grown at 30°C. Each panel shows representative results of an experiment repeated independently three times.
    Figure Legend Snippet: Adc17 Is Important for Proteasome Integrity (A) Native-PAGE (4.2%) of yeast extracts of wild-type (WT) or cim3-1 cultures, grown at 30°C or 37°C, revealed with the fluorogenic substrate Suc-LLVY-AMC. RP 2 CP, doubly capped proteasome; RPCP, singly capped proteasome. The alternative proteasome complex composed of the activator Bml10 bound to CP is indicated. Addition of 0.1% SDS activated the otherwise latent free CP. (B) Same as in (A) with cim3-1 cells transformed with the indicated plasmids or empty vector. RPT6 -complemented cells serve as a reference. (C) Native-PAGE of yeast extracts of cells of the indicated genotype, grown at 30°C. (D–F) Quantification of Rpt6 levels, normalized to control (either wild-type or Rpt6 complemented cells), are shown below Rpt6 blots. (D) Immunoblots of total extracts of wild-type or cim3-1 cells grown at 30°C or 37°C. (E) Same as in (D) with cim3-1 transformed with the indicated plasmids or empty vector. (F) Immunoblots of total extracts of cells of the indicated genotype grown at 30°C. Each panel shows representative results of an experiment repeated independently three times.

    Techniques Used: Clear Native PAGE, Transformation Assay, Plasmid Preparation, Western Blot

    5) Product Images from "The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation"

    Article Title: The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw045

    Nug1 nucleotide-binding mutant causes defects in 60S subunit biogenesis. ( A ) Growth behavior of Nug1 GTPase mutants were tested in a nug1 Δ background complemented by wild-type NUG 1 or mutant nug 1 K293A, T320A/T321A, D336N, G339A, N290D or N290L, all carried on a plasmid (YCplac22, CEN3, TRP1). Ten-fold serial dilutions of the indicated strains were spotted on YPD plates for 2 days at the indicated temperatures. Lower panel: whole cell lysates were prepared from exponentially growing cells for each of the indicated mutants. Samples were analyzed on SDS-PAGE and the protein levels of the Nug1 wild-type or mutants were determined by Western blotting using antibodies against the C-terminal TAP-tag (anti-protA). The Arc1 Western blot served as loading control and untagged Nug1 as negative control. ( B ) Top: Ribosome and polysome analysis of Nug1 wild-type and representative nucleotide-binding (D336N) and hydrolysis (G339A) mutants. Whole cell lysates were analyzed by sucrose gradient centrifugation. A 254nm profiles of the fractions collected are depicted. The red arrow denotes the increase of the 40S subunit and the red asterisks the half-mers. Bottom: Western blot of the gradient fractions using antibodies against Nug1 and Rpl12. ( C ) Rpl25-GFP localization in Nug1 wild-type and representative nucleotide-binding (D336N) and hydrolysis (G339A) mutants. ( D ) Northern hybridization and primer extension analysis (lower panel) of RNA extracted from Nug1 WT, nucleotide-binding (D336N) and Nug1 hydrolysis (G339A) mutants. Oligonucleotides used for hybridization or primer extension (P.E) are shown on the left of gel panels ( E ) Affinity-purification of Nug1 wild-type and nug 1 D336N or G339A mutants. ( F ) Affinity-purified Ssf1 pre-ribosomes in the presence of Nug1 wild-type or G-domain mutants (D336N, G339A). For (C) and (D) final eluates were analyzed by SDS-PAGE and Coomassie staining. The indicated bands were identified by mass spectrometry and/or by comparison with characterized TAP pre-ribosomal particles. Red asterisks (*) denote the baits. Black diamond (♦) corresponds to Rpl3, which was used as a loading control.
    Figure Legend Snippet: Nug1 nucleotide-binding mutant causes defects in 60S subunit biogenesis. ( A ) Growth behavior of Nug1 GTPase mutants were tested in a nug1 Δ background complemented by wild-type NUG 1 or mutant nug 1 K293A, T320A/T321A, D336N, G339A, N290D or N290L, all carried on a plasmid (YCplac22, CEN3, TRP1). Ten-fold serial dilutions of the indicated strains were spotted on YPD plates for 2 days at the indicated temperatures. Lower panel: whole cell lysates were prepared from exponentially growing cells for each of the indicated mutants. Samples were analyzed on SDS-PAGE and the protein levels of the Nug1 wild-type or mutants were determined by Western blotting using antibodies against the C-terminal TAP-tag (anti-protA). The Arc1 Western blot served as loading control and untagged Nug1 as negative control. ( B ) Top: Ribosome and polysome analysis of Nug1 wild-type and representative nucleotide-binding (D336N) and hydrolysis (G339A) mutants. Whole cell lysates were analyzed by sucrose gradient centrifugation. A 254nm profiles of the fractions collected are depicted. The red arrow denotes the increase of the 40S subunit and the red asterisks the half-mers. Bottom: Western blot of the gradient fractions using antibodies against Nug1 and Rpl12. ( C ) Rpl25-GFP localization in Nug1 wild-type and representative nucleotide-binding (D336N) and hydrolysis (G339A) mutants. ( D ) Northern hybridization and primer extension analysis (lower panel) of RNA extracted from Nug1 WT, nucleotide-binding (D336N) and Nug1 hydrolysis (G339A) mutants. Oligonucleotides used for hybridization or primer extension (P.E) are shown on the left of gel panels ( E ) Affinity-purification of Nug1 wild-type and nug 1 D336N or G339A mutants. ( F ) Affinity-purified Ssf1 pre-ribosomes in the presence of Nug1 wild-type or G-domain mutants (D336N, G339A). For (C) and (D) final eluates were analyzed by SDS-PAGE and Coomassie staining. The indicated bands were identified by mass spectrometry and/or by comparison with characterized TAP pre-ribosomal particles. Red asterisks (*) denote the baits. Black diamond (♦) corresponds to Rpl3, which was used as a loading control.

    Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation, SDS Page, Western Blot, Negative Control, Gradient Centrifugation, Northern Blot, Hybridization, Affinity Purification, Staining, Mass Spectrometry

    Nug1 depletion inhibits cell growth and causes defects in 60S subunit maturation. ( A ) The auxin-inducible degron system targets proteins for proteasomal degradation. Auxin (indole-3-acetic acid; IAA) induces degradation by mediating the interaction of the Aid-degron (fused to the protein target) with the substrate recognition domain of the TIR1 (F-box protein, auxin receptor). TIR is part of the SCF complex (E3 ubiquitin ligase) and leads to ubiquitination of the target and finally proteasomal degradation. ( B and C ) Depletion of Nug1 results in growth inhibition. Nug1 was genomically tagged at the C-terminal end with the Aid-tag. The ubiquitin E3 ligase TIR1 was genomically integrated and expressed under the constitutive ADH1 promoter ( P ADH 1 ). ( B ) Cells expressing the Nug1-Aid was treated with 0.5 mM auxin and samples were taken at different time points (t = 0, 10, 20, 30, 60, 90 and 120 min). Whole-cell lysates were analyzed by SDS-PAGE followed by Western blotting using an anti-Nug1 antibody. The Arc1 Western blot served as loading control. ( C ) Growth analysis of yeast cells expressing Aid-tagged or untagged Nug1 in the ADH1 :: TIR1 background. Cells were spoted in 10-fold serial dilutions on YPD plates with or without 0.5 mM auxin and incubated at 23°C and 30°C for 1 day. ( D ) Ssf1-TAP and Nsa1-TAP pre-ribosomes were affinity-purified from yeast cells expressing the fusion Nug1-Aid protein following treatment with auxin. TAP eluates were analyzed by SDS-PAGE followed by Coomassie staining. Rpl3 (♦) was used as a loading control. Bait proteins are marked with a star (*). ( E ) Binding assays of Ct Nug1 and Ct Dbp10. FLAG- Ct Dbp10 was immobilized on anti-FLAG beads and full-length Ct Nug1 was added in 5- or 10-fold excess in the presence of E. coli lysate (EcL) to compete for unspecific binding. The bound material was eluted with loading buffer and analyzed by SDS-PAGE followed by Coomassie staining. As a negative control (mock), Ct Nug1 was incubated with anti-FLAG beads. The bands corresponding to Ct Dbp10 and Ct Nug1 are indicated with arrows and the IgG light chain is labeled by *.
    Figure Legend Snippet: Nug1 depletion inhibits cell growth and causes defects in 60S subunit maturation. ( A ) The auxin-inducible degron system targets proteins for proteasomal degradation. Auxin (indole-3-acetic acid; IAA) induces degradation by mediating the interaction of the Aid-degron (fused to the protein target) with the substrate recognition domain of the TIR1 (F-box protein, auxin receptor). TIR is part of the SCF complex (E3 ubiquitin ligase) and leads to ubiquitination of the target and finally proteasomal degradation. ( B and C ) Depletion of Nug1 results in growth inhibition. Nug1 was genomically tagged at the C-terminal end with the Aid-tag. The ubiquitin E3 ligase TIR1 was genomically integrated and expressed under the constitutive ADH1 promoter ( P ADH 1 ). ( B ) Cells expressing the Nug1-Aid was treated with 0.5 mM auxin and samples were taken at different time points (t = 0, 10, 20, 30, 60, 90 and 120 min). Whole-cell lysates were analyzed by SDS-PAGE followed by Western blotting using an anti-Nug1 antibody. The Arc1 Western blot served as loading control. ( C ) Growth analysis of yeast cells expressing Aid-tagged or untagged Nug1 in the ADH1 :: TIR1 background. Cells were spoted in 10-fold serial dilutions on YPD plates with or without 0.5 mM auxin and incubated at 23°C and 30°C for 1 day. ( D ) Ssf1-TAP and Nsa1-TAP pre-ribosomes were affinity-purified from yeast cells expressing the fusion Nug1-Aid protein following treatment with auxin. TAP eluates were analyzed by SDS-PAGE followed by Coomassie staining. Rpl3 (♦) was used as a loading control. Bait proteins are marked with a star (*). ( E ) Binding assays of Ct Nug1 and Ct Dbp10. FLAG- Ct Dbp10 was immobilized on anti-FLAG beads and full-length Ct Nug1 was added in 5- or 10-fold excess in the presence of E. coli lysate (EcL) to compete for unspecific binding. The bound material was eluted with loading buffer and analyzed by SDS-PAGE followed by Coomassie staining. As a negative control (mock), Ct Nug1 was incubated with anti-FLAG beads. The bands corresponding to Ct Dbp10 and Ct Nug1 are indicated with arrows and the IgG light chain is labeled by *.

    Techniques Used: Inhibition, Expressing, SDS Page, Western Blot, Incubation, Affinity Purification, Staining, Binding Assay, Negative Control, Labeling

    6) Product Images from "TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation"

    Article Title: TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation

    Journal: Molecular Vision

    doi:

    TNF-α induces ROS generation in human RPE cells by activating NADPH oxidase. A : Reactive oxygen species (ROS) generation measured with 2',7'-dichlorofluorescein diacetate (DCFDA) fluorescence in human RPE cells treated with recombinant human tumor necrosis factor alpha (TNF-α) at various concentrations for 30 min (***p
    Figure Legend Snippet: TNF-α induces ROS generation in human RPE cells by activating NADPH oxidase. A : Reactive oxygen species (ROS) generation measured with 2',7'-dichlorofluorescein diacetate (DCFDA) fluorescence in human RPE cells treated with recombinant human tumor necrosis factor alpha (TNF-α) at various concentrations for 30 min (***p

    Techniques Used: Fluorescence, Recombinant

    TNF-α-mediated NF-ҡB activation is not involved in VEGF upregulation in human RPE cells. A : Western blots of p-p65, total p65, and β-actin. B : Real-time PCR of vascular endothelial growth factor (VEGF) in RPE cells pretreated with Bay 11–7082 (5 µM) or control dimethyl sulfoxide (DMSO) for 30 min before incubation with tumor necrosis factor alpha (TNF-α; 20 ng/ml) for an additional ( A ) 5, 15, or 30 min or ( B ) 12 h (***p
    Figure Legend Snippet: TNF-α-mediated NF-ҡB activation is not involved in VEGF upregulation in human RPE cells. A : Western blots of p-p65, total p65, and β-actin. B : Real-time PCR of vascular endothelial growth factor (VEGF) in RPE cells pretreated with Bay 11–7082 (5 µM) or control dimethyl sulfoxide (DMSO) for 30 min before incubation with tumor necrosis factor alpha (TNF-α; 20 ng/ml) for an additional ( A ) 5, 15, or 30 min or ( B ) 12 h (***p

    Techniques Used: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Incubation

    TNF-α upregulates VEGF expression in RPE cells via ROS-dependent signaling. A : Western blot of vascular endothelial growth factor (VEGF) in RPE cells treated with tumor necrosis factor alpha (TNF-α; 20 ng/ml) for 3, 6, or 12 h. B : Quantitative PCR of VEGF mRNA in RPE cells pretreated with apocynin (100 µM) or control for 30 min before incubation with TNF-α (20 ng/ml) for an additional 12 h (***p
    Figure Legend Snippet: TNF-α upregulates VEGF expression in RPE cells via ROS-dependent signaling. A : Western blot of vascular endothelial growth factor (VEGF) in RPE cells treated with tumor necrosis factor alpha (TNF-α; 20 ng/ml) for 3, 6, or 12 h. B : Quantitative PCR of VEGF mRNA in RPE cells pretreated with apocynin (100 µM) or control for 30 min before incubation with TNF-α (20 ng/ml) for an additional 12 h (***p

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Incubation

    TNF-α upregulates VEGF by mediating ROS-dependent transcriptional activation of β-catenin. Representative western blots of ( A ) β-catenin and ( B ) vascular endothelial growth factor (VEGF) in RPE cells pretreated with JW67 (20 µM) or XAV939 (1 µM) or control dimethyl sulfoxide (DMSO) for 30 min before incubation with tumor necrosis factor alpha (TNF-α; 20 ng/ml) for an additional 1, 3, or 6 h. C : Coimmunoprecipitation of β-catenin and T cell factor 1 (TCF1) in RPE cells pretreated with apocynin (APO, 100 µM) or XAV939 or control DMSO for 30 min (representative blot). D : Coimmunoprecipitation of β-catenin and TCF1 in RPE cells transfected with p22phox siRNA and incubated with TNFα (20 ng/ml) for 2 h (representative blot).
    Figure Legend Snippet: TNF-α upregulates VEGF by mediating ROS-dependent transcriptional activation of β-catenin. Representative western blots of ( A ) β-catenin and ( B ) vascular endothelial growth factor (VEGF) in RPE cells pretreated with JW67 (20 µM) or XAV939 (1 µM) or control dimethyl sulfoxide (DMSO) for 30 min before incubation with tumor necrosis factor alpha (TNF-α; 20 ng/ml) for an additional 1, 3, or 6 h. C : Coimmunoprecipitation of β-catenin and T cell factor 1 (TCF1) in RPE cells pretreated with apocynin (APO, 100 µM) or XAV939 or control DMSO for 30 min (representative blot). D : Coimmunoprecipitation of β-catenin and TCF1 in RPE cells transfected with p22phox siRNA and incubated with TNFα (20 ng/ml) for 2 h (representative blot).

    Techniques Used: Activation Assay, Western Blot, Incubation, Transfection

    TNF-α mediates CNV formation in association with VEGF expression in a murine model of laser-induced CNV. A : Representative images of western blot of tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF) protein in RPE/choroids of C57Bl/6 6-week-old mice without laser treatment (non-lasered) or 7 days after laser treatment. B : Quantification of densitometry (lasered; *p
    Figure Legend Snippet: TNF-α mediates CNV formation in association with VEGF expression in a murine model of laser-induced CNV. A : Representative images of western blot of tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF) protein in RPE/choroids of C57Bl/6 6-week-old mice without laser treatment (non-lasered) or 7 days after laser treatment. B : Quantification of densitometry (lasered; *p

    Techniques Used: Expressing, Western Blot, Mouse Assay

    7) Product Images from "Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes"

    Article Title: Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes

    Journal: BMC Cancer

    doi: 10.1186/s12885-016-2355-5

    PRE promoter activity analysis by Dual luciferase assay. T47D, BT474, and MCF-7 cells were hormone-starved for 24 h and transfected with PRE-luc reporter plasmid along with phRl-TK Renilla control plasmid. The transfected T47D cells were treated with P4 ( a ), MPA ( b ), or R5020 ( c) ± TPA (10nM, 100nM, 1 μM) alone or in combination with E2 (1nM). The transfected MCF-7 ( d ) and BT474 cells ( e ) received P4 or MPA ± TPA (10nM, 100nM, 1 μM). Luciferase activity was quantified using the Dual- Luciferase Reporter Assay Kit. The relative PRE- luciferase activity was expressed as the ratio of the firefly luciferase/Renilla luciferase unit (RLU)
    Figure Legend Snippet: PRE promoter activity analysis by Dual luciferase assay. T47D, BT474, and MCF-7 cells were hormone-starved for 24 h and transfected with PRE-luc reporter plasmid along with phRl-TK Renilla control plasmid. The transfected T47D cells were treated with P4 ( a ), MPA ( b ), or R5020 ( c) ± TPA (10nM, 100nM, 1 μM) alone or in combination with E2 (1nM). The transfected MCF-7 ( d ) and BT474 cells ( e ) received P4 or MPA ± TPA (10nM, 100nM, 1 μM). Luciferase activity was quantified using the Dual- Luciferase Reporter Assay Kit. The relative PRE- luciferase activity was expressed as the ratio of the firefly luciferase/Renilla luciferase unit (RLU)

    Techniques Used: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Reporter Assay

    Analysis of gene expression microarray. T47D cells were treated with R5020 (10nM) ± TPA (1 μM) for 24 h. Vehicle treated cells were used as a control. Differential gene expression was assayed using the Illumina platform. ( a ) Heatmap of 589 genes commonly regulated by R5020 and TPA. ( b ) Identification of 16 cell cycle genes upregulated by progesterone both in normal and breast cancer cells. ( c ) Top ten enriched pathways for control vs. R5020 and R5020 vs. R5020 + TPA analyzed by GO. ( d ) Top ten enriched cell processes for control vs. R5020 and R5020 vs. R5020 + TPA
    Figure Legend Snippet: Analysis of gene expression microarray. T47D cells were treated with R5020 (10nM) ± TPA (1 μM) for 24 h. Vehicle treated cells were used as a control. Differential gene expression was assayed using the Illumina platform. ( a ) Heatmap of 589 genes commonly regulated by R5020 and TPA. ( b ) Identification of 16 cell cycle genes upregulated by progesterone both in normal and breast cancer cells. ( c ) Top ten enriched pathways for control vs. R5020 and R5020 vs. R5020 + TPA analyzed by GO. ( d ) Top ten enriched cell processes for control vs. R5020 and R5020 vs. R5020 + TPA

    Techniques Used: Expressing, Microarray

    RT-qPCR validation of array data. RT- qPCR data for the sixteen genes show is displayed as a heat map (low to high: yellow to red) with fold-change in mRNA expression within the boxes. Hormone-starved T47D and MCF10A cells were treated for 24 h with Progesterone (P4), Medroxyprogesterone acetate (MPA), or Promegestol (R5020) alone or in combination with telapristone actetate (TPA) as indicated above the map. There were six independent repeats of the experiment. */**/*** represent p-values of
    Figure Legend Snippet: RT-qPCR validation of array data. RT- qPCR data for the sixteen genes show is displayed as a heat map (low to high: yellow to red) with fold-change in mRNA expression within the boxes. Hormone-starved T47D and MCF10A cells were treated for 24 h with Progesterone (P4), Medroxyprogesterone acetate (MPA), or Promegestol (R5020) alone or in combination with telapristone actetate (TPA) as indicated above the map. There were six independent repeats of the experiment. */**/*** represent p-values of

    Techniques Used: Quantitative RT-PCR, Expressing

    Determination of cell viability by MTT assay. T47D cells were hormone-starved for 24 h and treated for 24, 48, and 72 h with ( a ) P4 ± TPA, ( b ) MPA ± TPA, ( c ) R5020 ± TPA alone, or in combination with E2 ( d, e , and f ). Cells were also treated with E2 ± TPA ( g ). Vehicle treated cells were used as a control. X-axis: 24, 48, and 72 h time points. p -values for the various comparisons are provided in Additional file 2 : Table S1
    Figure Legend Snippet: Determination of cell viability by MTT assay. T47D cells were hormone-starved for 24 h and treated for 24, 48, and 72 h with ( a ) P4 ± TPA, ( b ) MPA ± TPA, ( c ) R5020 ± TPA alone, or in combination with E2 ( d, e , and f ). Cells were also treated with E2 ± TPA ( g ). Vehicle treated cells were used as a control. X-axis: 24, 48, and 72 h time points. p -values for the various comparisons are provided in Additional file 2 : Table S1

    Techniques Used: MTT Assay

    Annexin V and Ki67 expression analysis by flow cytometry. T47D cells were serum-starved for 24 h and treated with R5020 ± TPA for 24, 48 and 72 h. The percent of cells expressing each of the proteins was determined using flow cytometry. a . Annexin V. b . Ki67. Vehicle-treated cells were used as a control. * represents p value
    Figure Legend Snippet: Annexin V and Ki67 expression analysis by flow cytometry. T47D cells were serum-starved for 24 h and treated with R5020 ± TPA for 24, 48 and 72 h. The percent of cells expressing each of the proteins was determined using flow cytometry. a . Annexin V. b . Ki67. Vehicle-treated cells were used as a control. * represents p value

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Cell cycle of T47D cells and BT474 cells after treatment with R5020 [10nM] or E2 [1nM] + R5020 [10nM] alone or in presence of TPA [1 μM]. a . T47D and b . BT474 cells were serum-starved for 24 h and subsequently treated with E2, R5020 and the antiprogestin TPA in various combination as indicated in figure for 24 h. Cell cycle analysis was performed in presence of Propidium Iodide to measure G1, S and G2/M fractions. c . Immunoblot of increased PR expression after 72 h of exposure of BT474 cells to E2 (left) and after 24 h of exposure to R5020 (right). E2 significantly increase both PR-A and B protein expression. The loss of PR expression with exposure to R5020 is indicative of high transcriptional activity and rapid protein turnover [ 44 ]. The blot has been cropped to remove the 48 h data. d . *BT474 cells were stimulated with E2 [1nM] for 72 h prior to treatment of R5020 and TPA to increase PR expression
    Figure Legend Snippet: Cell cycle of T47D cells and BT474 cells after treatment with R5020 [10nM] or E2 [1nM] + R5020 [10nM] alone or in presence of TPA [1 μM]. a . T47D and b . BT474 cells were serum-starved for 24 h and subsequently treated with E2, R5020 and the antiprogestin TPA in various combination as indicated in figure for 24 h. Cell cycle analysis was performed in presence of Propidium Iodide to measure G1, S and G2/M fractions. c . Immunoblot of increased PR expression after 72 h of exposure of BT474 cells to E2 (left) and after 24 h of exposure to R5020 (right). E2 significantly increase both PR-A and B protein expression. The loss of PR expression with exposure to R5020 is indicative of high transcriptional activity and rapid protein turnover [ 44 ]. The blot has been cropped to remove the 48 h data. d . *BT474 cells were stimulated with E2 [1nM] for 72 h prior to treatment of R5020 and TPA to increase PR expression

    Techniques Used: Cell Cycle Assay, Expressing, Activity Assay

    Cell cycle analysis by flow cytometry. T47D cells were hormone-starved for 24 h and treated with progestogens (P4, MPA, R5020) ± TPA ( a, b , and c ) and in combination with E2 ( d, e , and f ) for 24 h. The fraction of cells in G1, S and G2/M phase was determined by flow cytometry using Propidium iodide. Vehicle-treated cells were used as a control
    Figure Legend Snippet: Cell cycle analysis by flow cytometry. T47D cells were hormone-starved for 24 h and treated with progestogens (P4, MPA, R5020) ± TPA ( a, b , and c ) and in combination with E2 ( d, e , and f ) for 24 h. The fraction of cells in G1, S and G2/M phase was determined by flow cytometry using Propidium iodide. Vehicle-treated cells were used as a control

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Cytometry

    8) Product Images from "Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation"

    Article Title: Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0164053

    SDS-PAGE, Western Blotting and SEC profiles of the Drug substances PfAMA1 DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3. Panel A: Reducing SDS-PAGE, silver-stained. Panel B: Non-reducing SDS-PAGE, silver-stained. Panel C: Non-reducing SDS-PAGE and Western-Blot, immune-staining using the reduction-sensitive monoclonal anti-PfAMA1 antibody 4G2. Panel D: SE-Chromatography of each of the PfAMA1 Drug Substances. PfAMA1 DiCo1 (black), PfAMA1 DiCo2 (blue) and PfAMA1 DiCo3 (red) was carried out on a Superdex200 10/300 GL prepacked column (GE Healthcare), using an Åkta Purifier Basic (GE Healthcare). PBS (140 mM NaCl, 10 mM sodium phosphate, 3 mM KCl, pH 7.4, Calbiochem) was used as running buffer at 0.5 mL.min -1 .
    Figure Legend Snippet: SDS-PAGE, Western Blotting and SEC profiles of the Drug substances PfAMA1 DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3. Panel A: Reducing SDS-PAGE, silver-stained. Panel B: Non-reducing SDS-PAGE, silver-stained. Panel C: Non-reducing SDS-PAGE and Western-Blot, immune-staining using the reduction-sensitive monoclonal anti-PfAMA1 antibody 4G2. Panel D: SE-Chromatography of each of the PfAMA1 Drug Substances. PfAMA1 DiCo1 (black), PfAMA1 DiCo2 (blue) and PfAMA1 DiCo3 (red) was carried out on a Superdex200 10/300 GL prepacked column (GE Healthcare), using an Åkta Purifier Basic (GE Healthcare). PBS (140 mM NaCl, 10 mM sodium phosphate, 3 mM KCl, pH 7.4, Calbiochem) was used as running buffer at 0.5 mL.min -1 .

    Techniques Used: SDS Page, Western Blot, Size-exclusion Chromatography, Staining, Chromatography

    Signature peptide MS analysis of pre-GMP produced PfAMA1 DiCo Drug substance intermediates. Panel A. Overloaded SDS-Gel of HIC eluate fractions with excised bands in marked boxes. Panel B. Examples of signature peptides identified in the 21 excised protein bands for PfAMA1-DiCo preparations. Key to preparations: PfAMA1 DiCo1 (Green), PfAMA1 DiCo2 (Orange) and PfAMA1 DiCo3 (Blue). In yellow the signature amino acids in these peptides, allowing for the differentiation between individual PfAMA1 DiCo proteins. Numbering of the amino acids is as in [ 15 ].
    Figure Legend Snippet: Signature peptide MS analysis of pre-GMP produced PfAMA1 DiCo Drug substance intermediates. Panel A. Overloaded SDS-Gel of HIC eluate fractions with excised bands in marked boxes. Panel B. Examples of signature peptides identified in the 21 excised protein bands for PfAMA1-DiCo preparations. Key to preparations: PfAMA1 DiCo1 (Green), PfAMA1 DiCo2 (Orange) and PfAMA1 DiCo3 (Blue). In yellow the signature amino acids in these peptides, allowing for the differentiation between individual PfAMA1 DiCo proteins. Numbering of the amino acids is as in [ 15 ].

    Techniques Used: Mass Spectrometry, Produced, SDS-Gel, Hydrophobic Interaction Chromatography

    Short time stability of the Drug Product in the presence of adjuvants. Drug Product (DP) was formulated with either Alhydrogel (alum) or GLA-SE. Formulae were broken either directly after formulation (0 hours) or after 24 hours of storage at 4°C or at 25°C. Samples were analysed with SDS-PAGE, both reduced/silver staining (panel A) and non-reduced/silver staining (panel B) and by (non-reduced) western-blot with the monoclonal antibody 4G2 (panel C).
    Figure Legend Snippet: Short time stability of the Drug Product in the presence of adjuvants. Drug Product (DP) was formulated with either Alhydrogel (alum) or GLA-SE. Formulae were broken either directly after formulation (0 hours) or after 24 hours of storage at 4°C or at 25°C. Samples were analysed with SDS-PAGE, both reduced/silver staining (panel A) and non-reduced/silver staining (panel B) and by (non-reduced) western-blot with the monoclonal antibody 4G2 (panel C).

    Techniques Used: SDS Page, Silver Staining, Western Blot

    9) Product Images from "Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer MembranesA New View of the Lethal Apoptotic Pore"

    Article Title: Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer MembranesA New View of the Lethal Apoptotic Pore

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001394

    Mitochondrial outer membranes isolated from rat liver form vesicles that are essentially devoid of the mitochondrial inner membrane. Negatively stained outer membrane vesicles (OMVs) were visualized by electron microscopy (A). The purity of OMVs was evaluated by Western blot analysis (B) and by measurements of enzyme activities (C) of marker proteins: VDAC and monoaminooxidase (MAO) for the mitochondrial outer membrane (MOM); cytochrome c oxidase subunit (COXIV) and succinate dehydrogenase (SDH) for the inner membrane; and calnexin for the ER. Shown in (B) are mitochondria (MITO) and two preparations of MOM (upper panel); ER and light mitochondrial fraction (ER+MITO) in comparison with two preparations of mitochondria, mitoplasts (MPL), and MOM (lower panel). Samples were loaded at 2.5 µg of protein or as indicated. (C) Specific SDH and MAO activities in MOM and mitoplasts relative to the corresponding activities in whole mitochondria. Activities were measured in four to six preparations and data are presented as means ± S.E.
    Figure Legend Snippet: Mitochondrial outer membranes isolated from rat liver form vesicles that are essentially devoid of the mitochondrial inner membrane. Negatively stained outer membrane vesicles (OMVs) were visualized by electron microscopy (A). The purity of OMVs was evaluated by Western blot analysis (B) and by measurements of enzyme activities (C) of marker proteins: VDAC and monoaminooxidase (MAO) for the mitochondrial outer membrane (MOM); cytochrome c oxidase subunit (COXIV) and succinate dehydrogenase (SDH) for the inner membrane; and calnexin for the ER. Shown in (B) are mitochondria (MITO) and two preparations of MOM (upper panel); ER and light mitochondrial fraction (ER+MITO) in comparison with two preparations of mitochondria, mitoplasts (MPL), and MOM (lower panel). Samples were loaded at 2.5 µg of protein or as indicated. (C) Specific SDH and MAO activities in MOM and mitoplasts relative to the corresponding activities in whole mitochondria. Activities were measured in four to six preparations and data are presented as means ± S.E.

    Techniques Used: Isolation, Staining, Electron Microscopy, Western Blot, Marker

    10) Product Images from "Vigilin interacts with signal peptide peptidase"

    Article Title: Vigilin interacts with signal peptide peptidase

    Journal: Proteome Science

    doi: 10.1186/1477-5956-10-33

    SPP exists in higher molecular weight complexes. Top panel , HEK293 lysate solubilized in 0.5% DDM and resolved on 16% Bis-Tris Blue Native-PAGE gels, revealing SPP-containing complexes at around 450 kDa, 200 kDa and 100 kDa. Lower panel , lysates resolved on the BN-PAGE were then resolved on the second dimension/SDS-PAGE. SDS-stable SPP dimer can be found in all three high molecular weight bands suggesting that the 100 kDa band observed in the first dimension is a SDS-stable SPP dimer.
    Figure Legend Snippet: SPP exists in higher molecular weight complexes. Top panel , HEK293 lysate solubilized in 0.5% DDM and resolved on 16% Bis-Tris Blue Native-PAGE gels, revealing SPP-containing complexes at around 450 kDa, 200 kDa and 100 kDa. Lower panel , lysates resolved on the BN-PAGE were then resolved on the second dimension/SDS-PAGE. SDS-stable SPP dimer can be found in all three high molecular weight bands suggesting that the 100 kDa band observed in the first dimension is a SDS-stable SPP dimer.

    Techniques Used: Molecular Weight, Blue Native PAGE, Polyacrylamide Gel Electrophoresis, SDS Page

    Vigilin is part of the 450 kDa SPP complex. ( A ) HEK293 cell lysate were solubilized in 0.5% DDM and resolved on 4-16% BN-PAGE gels. As shown on the left panel, SPP formed three distinct complexes at 450 kDa, 200 kDa and 100 kDa. Lysates probed with anti-vigilin antibody reveals that endogenous vigilin only forms one distinct complex on the BN-PAGE and that band co-migrates with the 450 kDa SPP complex. Lysates were also Coomassie stained to show that the 450 kDa SPP and vigilin containing complex was not a compression artifact of BN-PAGE gels, such as the band migrating at 700 kDa. ( B ) HEK293 vigilin-FLAG cell lysate were solubilized in 0.5% DDM and resolved on 4-16% BN-PAGE gels reveal that vigilin-FLAG also co-migrates with the 450 kDa SPP complex only. Data shown are representative blots from three independent experiments.
    Figure Legend Snippet: Vigilin is part of the 450 kDa SPP complex. ( A ) HEK293 cell lysate were solubilized in 0.5% DDM and resolved on 4-16% BN-PAGE gels. As shown on the left panel, SPP formed three distinct complexes at 450 kDa, 200 kDa and 100 kDa. Lysates probed with anti-vigilin antibody reveals that endogenous vigilin only forms one distinct complex on the BN-PAGE and that band co-migrates with the 450 kDa SPP complex. Lysates were also Coomassie stained to show that the 450 kDa SPP and vigilin containing complex was not a compression artifact of BN-PAGE gels, such as the band migrating at 700 kDa. ( B ) HEK293 vigilin-FLAG cell lysate were solubilized in 0.5% DDM and resolved on 4-16% BN-PAGE gels reveal that vigilin-FLAG also co-migrates with the 450 kDa SPP complex only. Data shown are representative blots from three independent experiments.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining

    11) Product Images from "Size-independent symmetric division in extraordinarily long cells"

    Article Title: Size-independent symmetric division in extraordinarily long cells

    Journal: Nature Communications

    doi: 10.1038/ncomms5803

    FISH/laser-scanning confocal microscopy (LSCM) of ectosymbionts attached to the worm surface. Images of the Efs coat are shown in a – e and images of the Eds coat in f – j . a and f are the corresponding bright field images of b – e and g – j , respectively. Each single symbiont is triple stained with specific probes targeting Eubacteria ( b and g ), Gammaproteobacteria ( c and h ), and the symbiont (Efs1027 and Eds214, respectively; d and i ). ( e ) and ( j ) are overlay pictures of ( b – d ) and ( g – i ), respectively. A single Efs cell is shown in the insets in ( a – e ). Scale bar, 25 μm for all images and 5 μm for the insert.
    Figure Legend Snippet: FISH/laser-scanning confocal microscopy (LSCM) of ectosymbionts attached to the worm surface. Images of the Efs coat are shown in a – e and images of the Eds coat in f – j . a and f are the corresponding bright field images of b – e and g – j , respectively. Each single symbiont is triple stained with specific probes targeting Eubacteria ( b and g ), Gammaproteobacteria ( c and h ), and the symbiont (Efs1027 and Eds214, respectively; d and i ). ( e ) and ( j ) are overlay pictures of ( b – d ) and ( g – i ), respectively. A single Efs cell is shown in the insets in ( a – e ). Scale bar, 25 μm for all images and 5 μm for the insert.

    Techniques Used: Fluorescence In Situ Hybridization, Confocal Microscopy, Staining

    12) Product Images from "Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)"

    Article Title: Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

    Journal: Current Chemical Genomics

    doi: 10.2174/1875397301105010030

    Characterization of GST-GAPDHS. ( A ) SDS-PAGE analysis of purified recombinant human GST–GAPDHS. GST-GAPDHS was expressed in the gapA-deficient E. coli strain DS112 and purified using glutathione-Sepharose. Coomassie-Blue stained 4-12% Bis-Tris gel, lane M, Prestained protein markers (Novex); lane 1, recombinant human GST-GAPDHS (2 µg). ( B ) Size exclusion chromatography of purified GST-GAPDHS. Purified human GST-GAPDHS was loaded on a Superose 12 column at 0.5 ml/min in 50 mM potassium phosphate pH 7.0, 400 mM NaCl buffer. The molecular size of the peaks indicated by the numbers were estimated as described previously [ 22 ].
    Figure Legend Snippet: Characterization of GST-GAPDHS. ( A ) SDS-PAGE analysis of purified recombinant human GST–GAPDHS. GST-GAPDHS was expressed in the gapA-deficient E. coli strain DS112 and purified using glutathione-Sepharose. Coomassie-Blue stained 4-12% Bis-Tris gel, lane M, Prestained protein markers (Novex); lane 1, recombinant human GST-GAPDHS (2 µg). ( B ) Size exclusion chromatography of purified GST-GAPDHS. Purified human GST-GAPDHS was loaded on a Superose 12 column at 0.5 ml/min in 50 mM potassium phosphate pH 7.0, 400 mM NaCl buffer. The molecular size of the peaks indicated by the numbers were estimated as described previously [ 22 ].

    Techniques Used: SDS Page, Purification, Recombinant, Staining, Size-exclusion Chromatography

    13) Product Images from "Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase"

    Article Title: Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-018-1174-x

    Oxr1 modulates Gpi1 oligomerisation. a Dimerisation of Gpi1 in cells co-transfected with Gpi1 and either an empty vector or full-length (Oxr1-FL) or short (Oxr1-C) Oxr1 isoforms. Cells were treated with a cross-linker (DSP) and proteins were extracted in PBS; the loading buffer did not contain any reducing β-mercaptoethanol and samples were not boiled (non-reducing conditions). As a control, protein extracts from cells treated with DSP were incubated with the reducing agent β-mercaptoethanol and boiled (DSPβ/b). Vinculin (Vin) levels were used to control for equivalent loading. b – c Quantification of the dimeric ( b ) or tetrameric ( c ) versus monomeric forms of Gpi1 ( N = 6 independent repeats). d – e Western blot and quantification showing Gpi1 oligomerization in cerebellum from Oxr1 d/d and Oxr1 +/+ mice from proteins extracted in PBS and non-reducing conditions. Ponceau staining was used to control for equal loading. As a control, protein extracts from the same preparations were incubated with the reducing agent β-mercaptoethanol and boiled (β/b). α-Tubulin levels were used to control for equivalent loading ( N = 8 animals per group). f mRNA expression levels of Gpi1 in the cerebellum of Oxr1 +/+ or Oxr1 d/d mice by qRT-PCR ( N = 4 animals per group). g Gpi1 activity in N2a cells transfected with the vectors indicated compared to an empty vector control ( N = 3–5 independent repeats). h Gpi1 activity in N2a cells co-transfected with Gpi1 and either Oxr1-FL or Oxr1-C ( N = 3 independent repeats). Panels b , c , g , h : one-way ANOVA; Panels e , f: t-test; * p
    Figure Legend Snippet: Oxr1 modulates Gpi1 oligomerisation. a Dimerisation of Gpi1 in cells co-transfected with Gpi1 and either an empty vector or full-length (Oxr1-FL) or short (Oxr1-C) Oxr1 isoforms. Cells were treated with a cross-linker (DSP) and proteins were extracted in PBS; the loading buffer did not contain any reducing β-mercaptoethanol and samples were not boiled (non-reducing conditions). As a control, protein extracts from cells treated with DSP were incubated with the reducing agent β-mercaptoethanol and boiled (DSPβ/b). Vinculin (Vin) levels were used to control for equivalent loading. b – c Quantification of the dimeric ( b ) or tetrameric ( c ) versus monomeric forms of Gpi1 ( N = 6 independent repeats). d – e Western blot and quantification showing Gpi1 oligomerization in cerebellum from Oxr1 d/d and Oxr1 +/+ mice from proteins extracted in PBS and non-reducing conditions. Ponceau staining was used to control for equal loading. As a control, protein extracts from the same preparations were incubated with the reducing agent β-mercaptoethanol and boiled (β/b). α-Tubulin levels were used to control for equivalent loading ( N = 8 animals per group). f mRNA expression levels of Gpi1 in the cerebellum of Oxr1 +/+ or Oxr1 d/d mice by qRT-PCR ( N = 4 animals per group). g Gpi1 activity in N2a cells transfected with the vectors indicated compared to an empty vector control ( N = 3–5 independent repeats). h Gpi1 activity in N2a cells co-transfected with Gpi1 and either Oxr1-FL or Oxr1-C ( N = 3 independent repeats). Panels b , c , g , h : one-way ANOVA; Panels e , f: t-test; * p

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Western Blot, Mouse Assay, Staining, Expressing, Quantitative RT-PCR, Activity Assay

    14) Product Images from "Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization ▿"

    Article Title: Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00512-10

    Glycosylation patterns of E3-10.9K orthologs. C-terminally EGFP-tagged E3-4.8K, E3-7.7K, E3-9K, and E3-10.9K were expressed under tetracycline regulation in HeLa T-REx cells. Total protein was analyzed by SDS gel electrophoresis of NuPAGE Novex 12%
    Figure Legend Snippet: Glycosylation patterns of E3-10.9K orthologs. C-terminally EGFP-tagged E3-4.8K, E3-7.7K, E3-9K, and E3-10.9K were expressed under tetracycline regulation in HeLa T-REx cells. Total protein was analyzed by SDS gel electrophoresis of NuPAGE Novex 12%

    Techniques Used: SDS-Gel, Electrophoresis

    15) Product Images from "A gene expression signature classifying telomerase and ALT immortalisation reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT"

    Article Title: A gene expression signature classifying telomerase and ALT immortalisation reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT

    Journal: Oncogene

    doi: 10.1038/onc.2009.238

    The TERT regulatory network is shown at the protein level and predicted c-MYC activity is confirmed as significantly lower in ALT. (a) Western blotting shows protein level differences in 3 molecules of the 297 gene network. 15 μg of cell extracts were run on NuPAGE 4-12% Bis-Tris gels, transferred to Millipore nitrocellulose membrane and probed with appropriate antibodies. Blots were then stripped and reprobed with ERK1 loading control. Panels shown are representative panels of 2 separate blots. (b) c-Myc activity ELISA shows significantly lower activity in ALT cells. Interval plot shows the average of 6 ALT cell lines (WI38-SV40, KMST6, SKLU, SUSM1, SAOS and U2OS) and 4 telomerase cell lines (A2780, C33a, HT1080 and 5637) on 3 separate occasions with 4 replicates of each cell line. Crosshairs show mean expression for each group and error bars show 95% confidence intervals of the mean. T-test of the results were T-Value = −2.51 P-Value = 0.015 DF = 51.
    Figure Legend Snippet: The TERT regulatory network is shown at the protein level and predicted c-MYC activity is confirmed as significantly lower in ALT. (a) Western blotting shows protein level differences in 3 molecules of the 297 gene network. 15 μg of cell extracts were run on NuPAGE 4-12% Bis-Tris gels, transferred to Millipore nitrocellulose membrane and probed with appropriate antibodies. Blots were then stripped and reprobed with ERK1 loading control. Panels shown are representative panels of 2 separate blots. (b) c-Myc activity ELISA shows significantly lower activity in ALT cells. Interval plot shows the average of 6 ALT cell lines (WI38-SV40, KMST6, SKLU, SUSM1, SAOS and U2OS) and 4 telomerase cell lines (A2780, C33a, HT1080 and 5637) on 3 separate occasions with 4 replicates of each cell line. Crosshairs show mean expression for each group and error bars show 95% confidence intervals of the mean. T-test of the results were T-Value = −2.51 P-Value = 0.015 DF = 51.

    Techniques Used: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

    16) Product Images from "Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest"

    Article Title: Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.211

    DDR/DNA repair proteins show suboptimal activation in TICS compared with bulk cells. ( a ) Western blot analysis of H125 bulk cells and TICs, 1, 4 and 24 h after IR, and A549, H1299 and H23 2 h after IR with 8 Gy. Phosphorylated and total forms of DNA-PK, ATM and phosphorylated forms of the ATM substrates H2AX, KAP1 and Chk2 were analyzed. α -Tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls. ( b ) Western blot analysis of H125 bulk cells and TICs, 4 h after bleomycin (2.5 μg/ml) or 24 h after cisplatin (1 or 10 μ M) exposure. Phosphorylated and total forms of DNA-PK, ATM, phosphorylated forms of the ATM substrates H2AX, KAP1, SMC1, Chk2 as well as the levels of FANCD2 and Rad51 were analyzed. A representative blot of at least two independent experiments is shown. β -Tubulin or GAPDH was used as loading controls. ( c ) Western blot analysis of H125 and A549 bulk cells and TICs, 24 h after 8 Gy IR, or 1 or 10 μ M continuous cisplatin exposure. FANCD2 and cdc25A was assayed. A representative blot of two independent experiments is shown. GAPDH was used as loading control. ( d ) Western blot analysis of PARP full-length and cleavage fragment 24 h after 10 μ M of continuous cisplatin treatment, with or without 1 h pretreatment with DNA-PKcs (NU7026) or ATM (KU55933) inhibitors (both 10 μ M). A representative blot of two independent experiments is shown. GAPDH was used as a loading control
    Figure Legend Snippet: DDR/DNA repair proteins show suboptimal activation in TICS compared with bulk cells. ( a ) Western blot analysis of H125 bulk cells and TICs, 1, 4 and 24 h after IR, and A549, H1299 and H23 2 h after IR with 8 Gy. Phosphorylated and total forms of DNA-PK, ATM and phosphorylated forms of the ATM substrates H2AX, KAP1 and Chk2 were analyzed. α -Tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls. ( b ) Western blot analysis of H125 bulk cells and TICs, 4 h after bleomycin (2.5 μg/ml) or 24 h after cisplatin (1 or 10 μ M) exposure. Phosphorylated and total forms of DNA-PK, ATM, phosphorylated forms of the ATM substrates H2AX, KAP1, SMC1, Chk2 as well as the levels of FANCD2 and Rad51 were analyzed. A representative blot of at least two independent experiments is shown. β -Tubulin or GAPDH was used as loading controls. ( c ) Western blot analysis of H125 and A549 bulk cells and TICs, 24 h after 8 Gy IR, or 1 or 10 μ M continuous cisplatin exposure. FANCD2 and cdc25A was assayed. A representative blot of two independent experiments is shown. GAPDH was used as loading control. ( d ) Western blot analysis of PARP full-length and cleavage fragment 24 h after 10 μ M of continuous cisplatin treatment, with or without 1 h pretreatment with DNA-PKcs (NU7026) or ATM (KU55933) inhibitors (both 10 μ M). A representative blot of two independent experiments is shown. GAPDH was used as a loading control

    Techniques Used: Activation Assay, Western Blot

    17) Product Images from "Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals"

    Article Title: Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014303

    Syx5 suppression reduces the amount of hCtr1 at the plasma membrane of Hek293 cells. Hek293 cells stably expressing Ctr1-myc were treated with scrambled negative control or Syx5 siRNA. Cell surface proteins were isolated following biotinylation using biotin-streptavidin precipitation prior to western immuno-blotting. Ctr1was detected with an anti-c-myc antibody. Syx5 knockdown was confirmed with an anti-Syx5 antibody and anti-NaK ATPase was used as control. Total lysate (T), non-biotinylated (NB), and biotinylated (B) fractions are shown. Protein bands were quantified with densitometry. Two species of Syx5 were detected and Syx5 knockdown reduced the amount of this protein in total cell lysate to approximately 33% of control siRNA treated cells. An hCtr1 monomer of approximately 35 kDa was detected in the biotinylated fraction. Densitometric analysis of hCtr1 protein intensity relative to NaK ATPase, revealed that Syx5 knockdown reduced the about of hCtr1 at the cell surface to approximately 20% of control siRNA treated cells.
    Figure Legend Snippet: Syx5 suppression reduces the amount of hCtr1 at the plasma membrane of Hek293 cells. Hek293 cells stably expressing Ctr1-myc were treated with scrambled negative control or Syx5 siRNA. Cell surface proteins were isolated following biotinylation using biotin-streptavidin precipitation prior to western immuno-blotting. Ctr1was detected with an anti-c-myc antibody. Syx5 knockdown was confirmed with an anti-Syx5 antibody and anti-NaK ATPase was used as control. Total lysate (T), non-biotinylated (NB), and biotinylated (B) fractions are shown. Protein bands were quantified with densitometry. Two species of Syx5 were detected and Syx5 knockdown reduced the amount of this protein in total cell lysate to approximately 33% of control siRNA treated cells. An hCtr1 monomer of approximately 35 kDa was detected in the biotinylated fraction. Densitometric analysis of hCtr1 protein intensity relative to NaK ATPase, revealed that Syx5 knockdown reduced the about of hCtr1 at the cell surface to approximately 20% of control siRNA treated cells.

    Techniques Used: Stable Transfection, Expressing, Negative Control, Isolation, Western Blot

    18) Product Images from "Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism"

    Article Title: Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171529

    Various immunostimulatory signals induce similar CV and R47H hTREM2 cleavage from the cell surface. (A) Representative flow cytometry plots demonstrating that both CV and R47H hTREM2 are lost from the cell surface of transduced RAW264.7 cells upon treatment with LPS, TNFα, or IFNγ. (B) Quantification of gMFI of LPS, TNFα, and IFNγ treated cells relative to untreated cells. Shown is mean ± SEM for three independent experiments. (C) Adam17 was knocked out in CV- and R47H-expressing RAW264.7 cells using lentivirally delivered Cas9 and guide RNA. LPS-stimulated cells were stained for hTREM2. Nontransduced cells (ctrl; gray shaded) uniformly lost hTREM2 surface expression. Transduced cells (solid red line) had two populations, one that retained hTREM2 surface expression (+) and one that lost hTREM2 surface expression (−), and these two populations were sorted by FACS. (D) Immunoblot for Adam17 demonstrates that the CV and R47H (+) populations have highly efficient knockout of Adam17 protein expression relative to either ctrl or (−) populations. (E) Experiments conducted as in A and B using Adam17 KO cells show that the effects of LPS, TNFα, and IFNγ on hTREM2 surface expression are ablated in Adam17-deficient cells. Shown is mean ± SEM for three independent experiments. **, P
    Figure Legend Snippet: Various immunostimulatory signals induce similar CV and R47H hTREM2 cleavage from the cell surface. (A) Representative flow cytometry plots demonstrating that both CV and R47H hTREM2 are lost from the cell surface of transduced RAW264.7 cells upon treatment with LPS, TNFα, or IFNγ. (B) Quantification of gMFI of LPS, TNFα, and IFNγ treated cells relative to untreated cells. Shown is mean ± SEM for three independent experiments. (C) Adam17 was knocked out in CV- and R47H-expressing RAW264.7 cells using lentivirally delivered Cas9 and guide RNA. LPS-stimulated cells were stained for hTREM2. Nontransduced cells (ctrl; gray shaded) uniformly lost hTREM2 surface expression. Transduced cells (solid red line) had two populations, one that retained hTREM2 surface expression (+) and one that lost hTREM2 surface expression (−), and these two populations were sorted by FACS. (D) Immunoblot for Adam17 demonstrates that the CV and R47H (+) populations have highly efficient knockout of Adam17 protein expression relative to either ctrl or (−) populations. (E) Experiments conducted as in A and B using Adam17 KO cells show that the effects of LPS, TNFα, and IFNγ on hTREM2 surface expression are ablated in Adam17-deficient cells. Shown is mean ± SEM for three independent experiments. **, P

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining, FACS, Knock-Out

    CV-KO-5XFAD mice show unique localization of soluble hTREM2 to neurons, plaques, and other nonmicroglial spaces. (A) Representative confocal images showing plaques (methoxy-X04, blue), microglia (Iba-1, red), and hTREM2 ECD (green) in the cortex. Bar, 30 µm. (B) Representative confocal images showing plaques (blue), hTREM2 C terminus (red), and hTREM2 ECD (green). In CV-KO-5XFAD brains, hTREM2 ECD is present in voxels that lack either Iba-1 or hTREM2 C terminus staining, whereas in R47H-KO-5XFAD, hTREM2 ECD largely colocalizes with Iba-1 and hTREM2 C terminus. Bar, 30 µm. (C and D) Mean intensity of hTREM2 ECD staining was quantified in Iba-1–positive (microglial) voxels and Iba-1–negative (nonmicroglial) voxels in (C) cortex and (D) hippocampus, showing similar expression levels in microglia but a significant increase outside of microglia in CV-KO-5XFAD brains only. (E) Representative confocal images showing plaques (methoxy-X04, blue), neuronal soma (NeuN, orange), and hTREM2 (green). Bar, 30 µm. (F) Frequency of hTREM2 + neurons and (G) mean hTREM2 intensity in neuronal soma was quantified in cortex and hippocampus. (H) Mean intensity of hTREM2 staining was quantified in methoxy-X04 + plaques in cortex and hippocampus. hTREM2 ECD staining on plaques and neuronal soma was significantly higher in CV-KO-5XFAD compared with R47H-KO-5XFAD. (I) Representative confocal image of plaques (blue), APP + dystrophic neurites (red), and hTREM2 ECD (green), showing a relative lack of soluble TREM2 on dystrophic neurites. Bar, 30 µm. (J) Representative confocal slice showing nuclei (ToPro-3, red) and hTREM2 ECD (green) within NeuN+ neuronal soma. hTREM2 + neurons do not show nuclear abnormalities characteristic of apoptosis. Bar, 10 µm. (K) Immunoblot of the PBS-soluble fraction of hippocampal homogenates for hTREM2, showing a hTREM2 smear at lower molecular weight than full-length hTREM2, indicating soluble TREM2. Densitometric analysis normalized to actin was performed, showing that CV-KO-5XFAD mice had more soluble TREM2 than CV-KO and R47H-KO-5XFAD mice. *, P
    Figure Legend Snippet: CV-KO-5XFAD mice show unique localization of soluble hTREM2 to neurons, plaques, and other nonmicroglial spaces. (A) Representative confocal images showing plaques (methoxy-X04, blue), microglia (Iba-1, red), and hTREM2 ECD (green) in the cortex. Bar, 30 µm. (B) Representative confocal images showing plaques (blue), hTREM2 C terminus (red), and hTREM2 ECD (green). In CV-KO-5XFAD brains, hTREM2 ECD is present in voxels that lack either Iba-1 or hTREM2 C terminus staining, whereas in R47H-KO-5XFAD, hTREM2 ECD largely colocalizes with Iba-1 and hTREM2 C terminus. Bar, 30 µm. (C and D) Mean intensity of hTREM2 ECD staining was quantified in Iba-1–positive (microglial) voxels and Iba-1–negative (nonmicroglial) voxels in (C) cortex and (D) hippocampus, showing similar expression levels in microglia but a significant increase outside of microglia in CV-KO-5XFAD brains only. (E) Representative confocal images showing plaques (methoxy-X04, blue), neuronal soma (NeuN, orange), and hTREM2 (green). Bar, 30 µm. (F) Frequency of hTREM2 + neurons and (G) mean hTREM2 intensity in neuronal soma was quantified in cortex and hippocampus. (H) Mean intensity of hTREM2 staining was quantified in methoxy-X04 + plaques in cortex and hippocampus. hTREM2 ECD staining on plaques and neuronal soma was significantly higher in CV-KO-5XFAD compared with R47H-KO-5XFAD. (I) Representative confocal image of plaques (blue), APP + dystrophic neurites (red), and hTREM2 ECD (green), showing a relative lack of soluble TREM2 on dystrophic neurites. Bar, 30 µm. (J) Representative confocal slice showing nuclei (ToPro-3, red) and hTREM2 ECD (green) within NeuN+ neuronal soma. hTREM2 + neurons do not show nuclear abnormalities characteristic of apoptosis. Bar, 10 µm. (K) Immunoblot of the PBS-soluble fraction of hippocampal homogenates for hTREM2, showing a hTREM2 smear at lower molecular weight than full-length hTREM2, indicating soluble TREM2. Densitometric analysis normalized to actin was performed, showing that CV-KO-5XFAD mice had more soluble TREM2 than CV-KO and R47H-KO-5XFAD mice. *, P

    Techniques Used: Mouse Assay, Staining, Expressing, Molecular Weight

    hTREM2 is expressed at comparable levels in microglia of CV and R47H transgenic mice. (A) Diagram showing generation of 5XFAD and non-5XFAD humanized TREM2 mice. (B) hTREM2 mRNA expression in KO, CV-KO, and R47H-KO whole cortical tissue. (C) Immunoblot of hTREM2 and actin in the PBS-insoluble fraction of hippocampal tissue homogenates shows similar relative hTREM2 protein abundance between CV-KO and R47H-KO by densitometric analysis normalized to actin. (D) Confocal microscopy of cortex of CV-KO, R47H-KO, or KO 5XFAD or non-5XFAD mice shows colocalization of hTREM2 C terminus (green) and microglial marker Iba-1 (red); methoxy-X04 staining for plaques is shown in blue. Bars, 50 µm for first three columns, 10 µm for last column. (E) Quantification of staining intensity of hTREM2 in the cortex and hippocampus. Staining intensity is similar between CV and R47H and increased in 5XFAD compared with non-5XFAD. *, P
    Figure Legend Snippet: hTREM2 is expressed at comparable levels in microglia of CV and R47H transgenic mice. (A) Diagram showing generation of 5XFAD and non-5XFAD humanized TREM2 mice. (B) hTREM2 mRNA expression in KO, CV-KO, and R47H-KO whole cortical tissue. (C) Immunoblot of hTREM2 and actin in the PBS-insoluble fraction of hippocampal tissue homogenates shows similar relative hTREM2 protein abundance between CV-KO and R47H-KO by densitometric analysis normalized to actin. (D) Confocal microscopy of cortex of CV-KO, R47H-KO, or KO 5XFAD or non-5XFAD mice shows colocalization of hTREM2 C terminus (green) and microglial marker Iba-1 (red); methoxy-X04 staining for plaques is shown in blue. Bars, 50 µm for first three columns, 10 µm for last column. (E) Quantification of staining intensity of hTREM2 in the cortex and hippocampus. Staining intensity is similar between CV and R47H and increased in 5XFAD compared with non-5XFAD. *, P

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Confocal Microscopy, Marker, Staining

    19) Product Images from "Reprogramming a Deubiquitinase into a Transamidase"

    Article Title: Reprogramming a Deubiquitinase into a Transamidase

    Journal: ACS chemical biology

    doi: 10.1021/acschembio.8b00759

    Autoubiquitination of evolved Yuh1 variants. (A) Structure shows the mutated residues in Yuh1qm5.6. (B) SDS-PAGE analysis of the autohydrolysis of mono-Ub-Yuh1qm variants. The monoubiquitinated products were generated from a Yuh1qm variant (1 μM) and Ub D77 (2 μM). Hydrolysis data were fit to a single exponential decay (Yuh1qm5.1, t 1/2 = 1400 ± 100 s; Yuh1qm5.3, t 1/2 = 500 ± 100 s; Yuh1qm5.6, t 1/2 = 800 ± 100 s). Error bars correspond to the standard error from three biological replicates. (C) MS/MS spectrum showing ubiquitination at K164 of Yuh1qm5.6.
    Figure Legend Snippet: Autoubiquitination of evolved Yuh1 variants. (A) Structure shows the mutated residues in Yuh1qm5.6. (B) SDS-PAGE analysis of the autohydrolysis of mono-Ub-Yuh1qm variants. The monoubiquitinated products were generated from a Yuh1qm variant (1 μM) and Ub D77 (2 μM). Hydrolysis data were fit to a single exponential decay (Yuh1qm5.1, t 1/2 = 1400 ± 100 s; Yuh1qm5.3, t 1/2 = 500 ± 100 s; Yuh1qm5.6, t 1/2 = 800 ± 100 s). Error bars correspond to the standard error from three biological replicates. (C) MS/MS spectrum showing ubiquitination at K164 of Yuh1qm5.6.

    Techniques Used: SDS Page, Generated, Variant Assay, Mass Spectrometry

    Autoubiquitination of Yuh1 variants. (A) SDS-PAGE analysis of autoubiquitination with Yuh1 variants (15 μ M) and Ub D77 (200 μ M). Bands 1−3 correspond to monoubiquitinated Yuh1 variants. (B) MS/MS spectrum showing ubiquitination at K66 of Yuh1qm. (C) SDS-PAGE analysis of Yuh1qm (5 μ M) autoubiquitination with Ub dimers (50 μ M). (D) MS/MS spectrum showing diubiquitination at K163 of Yuh1qm. (E) SDS-PAGE analysis of the autohydrolysis of monoubiquitinated Yuh1qm. The monoubiquitinated product (Ub-Yuh1qm) was generated from Yuh1qm (1 μ M) and Ub D77 (2 μ M). Hydrolysis data were fit to a single exponential decay (t 1/2 = 160 ± 60 s). Error bars correspond to the standard error from three biological replicates.
    Figure Legend Snippet: Autoubiquitination of Yuh1 variants. (A) SDS-PAGE analysis of autoubiquitination with Yuh1 variants (15 μ M) and Ub D77 (200 μ M). Bands 1−3 correspond to monoubiquitinated Yuh1 variants. (B) MS/MS spectrum showing ubiquitination at K66 of Yuh1qm. (C) SDS-PAGE analysis of Yuh1qm (5 μ M) autoubiquitination with Ub dimers (50 μ M). (D) MS/MS spectrum showing diubiquitination at K163 of Yuh1qm. (E) SDS-PAGE analysis of the autohydrolysis of monoubiquitinated Yuh1qm. The monoubiquitinated product (Ub-Yuh1qm) was generated from Yuh1qm (1 μ M) and Ub D77 (2 μ M). Hydrolysis data were fit to a single exponential decay (t 1/2 = 160 ± 60 s). Error bars correspond to the standard error from three biological replicates.

    Techniques Used: SDS Page, Mass Spectrometry, Generated

    20) Product Images from "Inhibition of Langerhans cell maturation by human papillomavirus type 16: a novel role for the annexin A2 heterotetramer in immune suppression"

    Article Title: Inhibition of Langerhans cell maturation by human papillomavirus type 16: a novel role for the annexin A2 heterotetramer in immune suppression

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1303190

    Downregulation of anxA2 reduces uptake of HPV16L1L2 VLP
    Figure Legend Snippet: Downregulation of anxA2 reduces uptake of HPV16L1L2 VLP

    Techniques Used:

    21) Product Images from "SOX11 expression correlates to promoter methylation and regulates tumor growth in hematopoietic malignancies"

    Article Title: SOX11 expression correlates to promoter methylation and regulates tumor growth in hematopoietic malignancies

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-187

    Overexpression of SOX11 decrease proliferation . a) mRNA expression of SOX11 at 24 h after overexpression of the SOX11 gene in six B cell lymphoma cell lines. b) Western blot analysis at 24 h confirms SOX11 overexpression in SOX11 transfected samples (right), compared to wt (left) and control vector (middle), loading control (GAPDH) is seen below c) Proliferation assay at 48 h after transfection showed decreased cell growth in all cell lines but BJAB where the decrease could be seen already after 24 h. In a) all values are relative quantity (RQ) compared to the GFP value for GRANTA 519. In c) all cell lines are compared to their respective GFP value. The data is representative of three independent assays. In a) the error bars show the 95% confidence interval, while in c) ±1SD is shown.
    Figure Legend Snippet: Overexpression of SOX11 decrease proliferation . a) mRNA expression of SOX11 at 24 h after overexpression of the SOX11 gene in six B cell lymphoma cell lines. b) Western blot analysis at 24 h confirms SOX11 overexpression in SOX11 transfected samples (right), compared to wt (left) and control vector (middle), loading control (GAPDH) is seen below c) Proliferation assay at 48 h after transfection showed decreased cell growth in all cell lines but BJAB where the decrease could be seen already after 24 h. In a) all values are relative quantity (RQ) compared to the GFP value for GRANTA 519. In c) all cell lines are compared to their respective GFP value. The data is representative of three independent assays. In a) the error bars show the 95% confidence interval, while in c) ±1SD is shown.

    Techniques Used: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Proliferation Assay

    Methylation status of SOX11 promoter region correlated to SOX11 expression . Methylation status of SOX11 promoter (described as percentage of methylated CpGs of 28 possible CpG methylation sites) was analyzed by direct bisulfite sequencing (right Y-axis) and correlated to SOX11 expression on mRNA (left Y-axis) and protein level in nineteen lymphoid or monocytic cell lines (Table 1). Generally, all samples with a ΔC T (SOX11+RT, SOX11-RT)
    Figure Legend Snippet: Methylation status of SOX11 promoter region correlated to SOX11 expression . Methylation status of SOX11 promoter (described as percentage of methylated CpGs of 28 possible CpG methylation sites) was analyzed by direct bisulfite sequencing (right Y-axis) and correlated to SOX11 expression on mRNA (left Y-axis) and protein level in nineteen lymphoid or monocytic cell lines (Table 1). Generally, all samples with a ΔC T (SOX11+RT, SOX11-RT)

    Techniques Used: Methylation, Expressing, CpG Methylation Assay, Methylation Sequencing

    SOX11 DNA methylation and protein expression in primary lymphoma samples . Methylation patterns of SOX11 promoter in clinical specimens was determined by bisulfite sequencing of individual alleles and correlated to SOX11 protein expression. Every row represents a unique allele and the columns represent a potentially methylated CpG site. a) In MCL samples, the promoter stays unmethylated and SOX11 is detectable. b) The lack of SOX11 protein in FL and DLBCL is accompanied by 50-100% methylated alleles.
    Figure Legend Snippet: SOX11 DNA methylation and protein expression in primary lymphoma samples . Methylation patterns of SOX11 promoter in clinical specimens was determined by bisulfite sequencing of individual alleles and correlated to SOX11 protein expression. Every row represents a unique allele and the columns represent a potentially methylated CpG site. a) In MCL samples, the promoter stays unmethylated and SOX11 is detectable. b) The lack of SOX11 protein in FL and DLBCL is accompanied by 50-100% methylated alleles.

    Techniques Used: DNA Methylation Assay, Expressing, Methylation, Methylation Sequencing

    siRNA knock-down of SOX11 increase proliferation . Effect of the siRNA induced knock-down of the SOX11 gene in GRANTA-519 and REC-1 on, a) mRNA level at 24 and 48 h; b) protein level at 48 h and 72 h, respectively, and c) proliferation at 24, 48 and 72 h. A control siRNA targeting the Eg5 gene was used as a positive control (only shown in b). All values in a) are relative quantity values (RQ) compared to the scrambled siRNA control. The data is representative of three independent assays. In a) the error bars show the 95% confidence interval, while in c) ±1 SD is shown.
    Figure Legend Snippet: siRNA knock-down of SOX11 increase proliferation . Effect of the siRNA induced knock-down of the SOX11 gene in GRANTA-519 and REC-1 on, a) mRNA level at 24 and 48 h; b) protein level at 48 h and 72 h, respectively, and c) proliferation at 24, 48 and 72 h. A control siRNA targeting the Eg5 gene was used as a positive control (only shown in b). All values in a) are relative quantity values (RQ) compared to the scrambled siRNA control. The data is representative of three independent assays. In a) the error bars show the 95% confidence interval, while in c) ±1 SD is shown.

    Techniques Used: Positive Control

    Gene Chip analysis reveal SOX11-induced regulation of Rb-E2F and TGF-β signaling pathways . Ingenuity Pathway Analysis identified the canonical pathway
    Figure Legend Snippet: Gene Chip analysis reveal SOX11-induced regulation of Rb-E2F and TGF-β signaling pathways . Ingenuity Pathway Analysis identified the canonical pathway "Molecular Mechanisms in Cancer" as highly associated with the 3647 deregulated genes. Within this pathway RB-E2F (A and B) and TGF-β (C and D) signaling is regulated in a time-dependent manner as shown after 24 h (A and C) and 48 h (B and D) of ectopic SOX11-overexpression. The differentially regulated genes are marked in red or green when the mean fold change for GRANTA-519 and JEKO-1 was ≥1.2 or ≤1.2. The remaining differentially regulated genes, including genes with different kinetics in the two cell lines, are marked in grey.

    Techniques Used: Chromatin Immunoprecipitation, Over Expression

    CpG islands in the SOX11 promoter region . Analysis of 2000 bp upstream of SOX11 transcription start revealed four CpG islands with a GC content above 50 percent http://www.urogene.org/methprimer/index1.html [ 45 ]. CpG dinucleotides are represented as vertical bars. Primers that amplified -435 to -222 were used in bisulfite sequencing to compare the methylation status of the SOX11 promoter region with SOX11 expression.
    Figure Legend Snippet: CpG islands in the SOX11 promoter region . Analysis of 2000 bp upstream of SOX11 transcription start revealed four CpG islands with a GC content above 50 percent http://www.urogene.org/methprimer/index1.html [ 45 ]. CpG dinucleotides are represented as vertical bars. Primers that amplified -435 to -222 were used in bisulfite sequencing to compare the methylation status of the SOX11 promoter region with SOX11 expression.

    Techniques Used: Amplification, Methylation Sequencing, Methylation, Expressing

    22) Product Images from "Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation"

    Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-44749-4

    mDia1 co-immunoprecipitates differentiation markers MyoD, active β-Catenin and pAkt2 Ser474 along with Phb2 during differentiation. C2C12 cells cultured under growth conditions (GM) or differentiated for 72 (D72) hours were lysed and subjected to IP with anti-mDia1 antibody, and analysed by western blotting using respective antibodies. ( a ) Co-IP of mDia1 with Akt2 and pAkt2 Ser474 in MT. IP samples were loaded on different gels, cut and processed in parallel for detection of mDia1, Akt2 and pAkt2 Ser474. ( b ) Co-IP of MyoD, Phb2 and Akt2 by mDia1 in MT. ( c ) Co-IP of active β-Catenin and Phb2 by mDia1 in MT. ( d ) IP of Phb1, Akt2 and Phb2 by mDia1 in MT. Act β-Cat- Active β-Catenin. For the blots shown in ( b – d ) the IP samples for MB and MT were run on a single gel, the blot was cut and processed in parallel for detection using the respective antibodies under the same conditions. Uncropped version of the blots represented in ( b – d ) are shown in Supplementary Fig. S6 .
    Figure Legend Snippet: mDia1 co-immunoprecipitates differentiation markers MyoD, active β-Catenin and pAkt2 Ser474 along with Phb2 during differentiation. C2C12 cells cultured under growth conditions (GM) or differentiated for 72 (D72) hours were lysed and subjected to IP with anti-mDia1 antibody, and analysed by western blotting using respective antibodies. ( a ) Co-IP of mDia1 with Akt2 and pAkt2 Ser474 in MT. IP samples were loaded on different gels, cut and processed in parallel for detection of mDia1, Akt2 and pAkt2 Ser474. ( b ) Co-IP of MyoD, Phb2 and Akt2 by mDia1 in MT. ( c ) Co-IP of active β-Catenin and Phb2 by mDia1 in MT. ( d ) IP of Phb1, Akt2 and Phb2 by mDia1 in MT. Act β-Cat- Active β-Catenin. For the blots shown in ( b – d ) the IP samples for MB and MT were run on a single gel, the blot was cut and processed in parallel for detection using the respective antibodies under the same conditions. Uncropped version of the blots represented in ( b – d ) are shown in Supplementary Fig. S6 .

    Techniques Used: Cell Culture, Western Blot, Co-Immunoprecipitation Assay

    mDia1 interacts with Phb2 in the cytoplasm of myotubes. ( a ) Immunostaining of endogenous mDia1 and Phb2 during proliferation (GM) and differentiation (D72) to detect colocalisation. The white boxes indicate the zoomed regions. Arrows indicate colocalised puncta. Confocal images were acquired using Leica TCS SP8 confocal microscope. ( b ) Purity of cytoplasmic and nuclear fractions of MT (D72). Cytoplasmic and nuclear extracts were prepared from D72 MT, followed by analysis by western blotting with antibodies against cytoplasmic GAPDH, nuclear LaminA/C and LaminB1 to determine the purity of the fractions. Distribution of mDia1 and Phb2 was detected by western blotting using respective antibodies. Lysates were loaded on a single gel, the blot was cut prior to processing in parallel for western blotting. ( c ) IP of mDia1 in cytoplasmic and nuclear extracts to detect localisation of associated Phb2. Cytoplasmic and nuclear extracts were prepared from D72 MT and subjected to IP using anti-mDia1 antibody, followed by detection of Phb2. IP products from cytoplasmic and nuclear extracts were loaded on a single gel, the blot was cut and probed for mDia1 and Phb2 in parallel. Cyt- Cytoplasm, Nucl-nucleus.
    Figure Legend Snippet: mDia1 interacts with Phb2 in the cytoplasm of myotubes. ( a ) Immunostaining of endogenous mDia1 and Phb2 during proliferation (GM) and differentiation (D72) to detect colocalisation. The white boxes indicate the zoomed regions. Arrows indicate colocalised puncta. Confocal images were acquired using Leica TCS SP8 confocal microscope. ( b ) Purity of cytoplasmic and nuclear fractions of MT (D72). Cytoplasmic and nuclear extracts were prepared from D72 MT, followed by analysis by western blotting with antibodies against cytoplasmic GAPDH, nuclear LaminA/C and LaminB1 to determine the purity of the fractions. Distribution of mDia1 and Phb2 was detected by western blotting using respective antibodies. Lysates were loaded on a single gel, the blot was cut prior to processing in parallel for western blotting. ( c ) IP of mDia1 in cytoplasmic and nuclear extracts to detect localisation of associated Phb2. Cytoplasmic and nuclear extracts were prepared from D72 MT and subjected to IP using anti-mDia1 antibody, followed by detection of Phb2. IP products from cytoplasmic and nuclear extracts were loaded on a single gel, the blot was cut and probed for mDia1 and Phb2 in parallel. Cyt- Cytoplasm, Nucl-nucleus.

    Techniques Used: Immunostaining, Microscopy, Western Blot

    Model: Phb2 sequesters mDia1 in the cytoplasmic puncta during differentiation to promote MyoG expression. mDia1 does not interact with Phb2 in MB and its expression decreases during differentiation. In MT, Phb2 binds and sequesters mDia1 in the cytoplasmic puncta, thereby restricting its availability and anti-myogenic activity to promote MyoG expression. The mDia1-Phb2 interaction might promote mitochondrial biogenesis in MT, thereby enhancing MyoG expression and differentiation. Although we have shown that mDia1 interacts with MyoD, pAkt2 Ser474, β-Catenin and Phb1, it remains unclear whether these interactors bind the cytoplasmic mDia1-Phb2 complex to regulate MyoG expression or exist as separate mDia1-interacting pools. Black dotted box indicates a possible mDia1 complex that might regulate MyoG in MT, but needs additional studies. Red dotted box highlights a cytoplasmic puncta. Dashed arrow highlights studies that need to be investigated.
    Figure Legend Snippet: Model: Phb2 sequesters mDia1 in the cytoplasmic puncta during differentiation to promote MyoG expression. mDia1 does not interact with Phb2 in MB and its expression decreases during differentiation. In MT, Phb2 binds and sequesters mDia1 in the cytoplasmic puncta, thereby restricting its availability and anti-myogenic activity to promote MyoG expression. The mDia1-Phb2 interaction might promote mitochondrial biogenesis in MT, thereby enhancing MyoG expression and differentiation. Although we have shown that mDia1 interacts with MyoD, pAkt2 Ser474, β-Catenin and Phb1, it remains unclear whether these interactors bind the cytoplasmic mDia1-Phb2 complex to regulate MyoG expression or exist as separate mDia1-interacting pools. Black dotted box indicates a possible mDia1 complex that might regulate MyoG in MT, but needs additional studies. Red dotted box highlights a cytoplasmic puncta. Dashed arrow highlights studies that need to be investigated.

    Techniques Used: Expressing, Activity Assay

    Mapping interaction domains on mDia1 and Phb2. ( a ) Schematic for mouse mDia1 truncation mutants. ( b ) Western blot to detect expression level of mDia1 mutants. Lysates from HEK293T transfected with mDia1 mutants were probed using anti-GFP antibody. GAPDH was used as a loading control. All the lysates were run on a single gel, the blot was cut and processed in parallel for detection of GFP-tagged mDia1 mutants and GAPDH. ( c , c ’) Co-IP of mDia1 mutants and Phb2 to map interaction domains. HEK293T cells were transfected with Phb2-Y2H and various mDia1 mutants, followed by IP with anti flag antibody. IP products were run on different gels, the blots were cut and processed in parallel for detection of interacting mutants. ( d ) Schematic for mouse Phb2 truncation mutants. ( e ) Western blot to detect expression of Phb2 mutants. Lysates of HEK293T transfected with Phb2 mutants were loaded on a single gel, the blot was cut and analysed in parallel using anti-flag and anti-GAPDH antibodies. GAPDH was used as a loading control. ( f ) Co-IP of Phb2 mutants and mDia1 to map interaction domains. Lysates from HEK293T cells co-transfected with various Phb2 mutants and mDia1ΔN3 were subjected to IP using anti-flag antibody. IP samples were run on different gels, the blots were cut and processed in parallel for detection with anti-GFP and anti-Flag antibodies under same conditions of detection. Different gels with 12% and 8% were used for the flag and GFP blots respectively. The represented cropped input lanes of Phb2-Amino, Central, Carboxy and 120–232 in the GFP blot show lower exposure of the inputs run along with the corresponding IP samples in the same gel whereas the cropped input lanes from Central, Carboxy and 120–232 in flag blot represent higher exposure of the inputs run along with the corresponding IP samples in the same gel. The numbers represent aa positions ( a , d ).
    Figure Legend Snippet: Mapping interaction domains on mDia1 and Phb2. ( a ) Schematic for mouse mDia1 truncation mutants. ( b ) Western blot to detect expression level of mDia1 mutants. Lysates from HEK293T transfected with mDia1 mutants were probed using anti-GFP antibody. GAPDH was used as a loading control. All the lysates were run on a single gel, the blot was cut and processed in parallel for detection of GFP-tagged mDia1 mutants and GAPDH. ( c , c ’) Co-IP of mDia1 mutants and Phb2 to map interaction domains. HEK293T cells were transfected with Phb2-Y2H and various mDia1 mutants, followed by IP with anti flag antibody. IP products were run on different gels, the blots were cut and processed in parallel for detection of interacting mutants. ( d ) Schematic for mouse Phb2 truncation mutants. ( e ) Western blot to detect expression of Phb2 mutants. Lysates of HEK293T transfected with Phb2 mutants were loaded on a single gel, the blot was cut and analysed in parallel using anti-flag and anti-GAPDH antibodies. GAPDH was used as a loading control. ( f ) Co-IP of Phb2 mutants and mDia1 to map interaction domains. Lysates from HEK293T cells co-transfected with various Phb2 mutants and mDia1ΔN3 were subjected to IP using anti-flag antibody. IP samples were run on different gels, the blots were cut and processed in parallel for detection with anti-GFP and anti-Flag antibodies under same conditions of detection. Different gels with 12% and 8% were used for the flag and GFP blots respectively. The represented cropped input lanes of Phb2-Amino, Central, Carboxy and 120–232 in the GFP blot show lower exposure of the inputs run along with the corresponding IP samples in the same gel whereas the cropped input lanes from Central, Carboxy and 120–232 in flag blot represent higher exposure of the inputs run along with the corresponding IP samples in the same gel. The numbers represent aa positions ( a , d ).

    Techniques Used: Western Blot, Expressing, Transfection, Co-Immunoprecipitation Assay

    Co-expression of mDia1 and Phb2 prevents repression of endogenous MyoG and MyoD. ( a) Overexpressed mDia1ΔN3 represses MyoG in MT, while co-expressed Phb2 reverses the repression. qRT-PCR analysis of MyoG transcripts in C2C12 transiently transfected with mDia1ΔN3 and Phb2 and shifted to DM for 36 hours. ***p
    Figure Legend Snippet: Co-expression of mDia1 and Phb2 prevents repression of endogenous MyoG and MyoD. ( a) Overexpressed mDia1ΔN3 represses MyoG in MT, while co-expressed Phb2 reverses the repression. qRT-PCR analysis of MyoG transcripts in C2C12 transiently transfected with mDia1ΔN3 and Phb2 and shifted to DM for 36 hours. ***p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    Prohibitin2, a novel mDia1-interacting protein, associates with mDia1 in myotubes. ( a ) Domain structure of full-length (FL) mDia1 and constitutively active mDia1 mutant, mDia1ΔN3. Grey lines indicate RhoA and DAD binding regions. G-GTPase binding domain, DID-Diaphanous Inhibitory Domain, Dimerisation Domain (DD), Coiled Coil (CC), FH1, FH2, FH3-Formin Homology domains, DAD-Diaphanous Auto-inhibitory Domain. Start positions of domains are depicted. ( b ) Phb2 identified as mDia1-interacting protein in a yeast two-hybrid screen. PJ69-4A was co-transformed with Phb2-AD and mDia1ΔN3-BD (positive GAL4 reconstitution) or empty-BD (negative GAL4 reconstitution) and four colonies per reconstitution were screened for ADE2 and LacZ reporters on −Trp/−Leu/−Ade and −Trp/−Leu + X-Gal plates respectively. Growth indicates ADE2 induction and blue pigmentation indicates LacZ induction. Positive control “P”- Drosophila Batman-AD and GAGA factor-BD, negative control “N”- empty-AD and empty-BD. Trp-Tryptophan, Leu-Leucine, Ade-Adenine. AD-Activation domain, BD-binding domain. ( c ) Domain structure of Phb2 FL and Phb2-Y2H. HYD-Hydrophobic region, PHB-Prohibitin domain, CC-Coiled coil domain. ( d ) Co-IP of flag-tagged Phb2-Y2H and GFP-tagged mDia1ΔN3 to confirm the interaction. HEK293T, co-transfected with mDia1ΔN3 and Phb2-Y2H, and pulled down with anti-Flag antibody. IP product was run on two different gels 8% and 12% for detecting with anti-GFP and anti-Flag antibodies respectively and these blots were processed in parallel. The blot probed with anti-Flag antibody represented here was cut prior to processing for western blotting. Cropped blot for GFP has been shown here whereas full-length GFP blot is presented in Supplementary Fig. S6 . ( e ) LC-MS/MS analysis of mDia1-interacting proteins in myoblasts (MB) and myotubes (MT) in differentiation medium (DM) for 72 hours. Venn diagram represents the number of proteins that bind mDia1 in MB or MT or both MB and MT. ( f ) Phb2 peptides identified in MT lysates by LC-MS/MS analysis of mDia1 IP proteins. Phb2 aa sequence (NCBI Reference Sequence # NP_031557.2) showing peptides identified in first (red), second and third (blue) and all three (underlined) biological replicates. ( g , h ) Reciprocal IP of endogenous mDia1 and Phb2 to identify stage-specific interaction. Lysates from proliferating MB (GM), MT in DM for 24 (D24) and 72 (D72) hours were harvested and subjected to IP with anti-mDia1 ( g ) or anti-Phb2 ( h ) antibodies. IP samples were loaded on different gels, blots were cut and processed in parallel, using same conditions of antibody incubation and exposure time during developing. ( i ) Western blot showing the expression profile of mDia1 and Phb2 in GM, D24, and D72 lysates. All the lysates were run on a single gel, blots were cut and probed for mDia1, Phb2, Akt2, MyoD, β-actin and GAPDH. The same lysates were run on a different gel, blots were cut and probed for MyoG, Akt1 and GAPDH. ( j , k ) Bar diagram represents the densitometric quantification of western blots shown in (i) *p
    Figure Legend Snippet: Prohibitin2, a novel mDia1-interacting protein, associates with mDia1 in myotubes. ( a ) Domain structure of full-length (FL) mDia1 and constitutively active mDia1 mutant, mDia1ΔN3. Grey lines indicate RhoA and DAD binding regions. G-GTPase binding domain, DID-Diaphanous Inhibitory Domain, Dimerisation Domain (DD), Coiled Coil (CC), FH1, FH2, FH3-Formin Homology domains, DAD-Diaphanous Auto-inhibitory Domain. Start positions of domains are depicted. ( b ) Phb2 identified as mDia1-interacting protein in a yeast two-hybrid screen. PJ69-4A was co-transformed with Phb2-AD and mDia1ΔN3-BD (positive GAL4 reconstitution) or empty-BD (negative GAL4 reconstitution) and four colonies per reconstitution were screened for ADE2 and LacZ reporters on −Trp/−Leu/−Ade and −Trp/−Leu + X-Gal plates respectively. Growth indicates ADE2 induction and blue pigmentation indicates LacZ induction. Positive control “P”- Drosophila Batman-AD and GAGA factor-BD, negative control “N”- empty-AD and empty-BD. Trp-Tryptophan, Leu-Leucine, Ade-Adenine. AD-Activation domain, BD-binding domain. ( c ) Domain structure of Phb2 FL and Phb2-Y2H. HYD-Hydrophobic region, PHB-Prohibitin domain, CC-Coiled coil domain. ( d ) Co-IP of flag-tagged Phb2-Y2H and GFP-tagged mDia1ΔN3 to confirm the interaction. HEK293T, co-transfected with mDia1ΔN3 and Phb2-Y2H, and pulled down with anti-Flag antibody. IP product was run on two different gels 8% and 12% for detecting with anti-GFP and anti-Flag antibodies respectively and these blots were processed in parallel. The blot probed with anti-Flag antibody represented here was cut prior to processing for western blotting. Cropped blot for GFP has been shown here whereas full-length GFP blot is presented in Supplementary Fig. S6 . ( e ) LC-MS/MS analysis of mDia1-interacting proteins in myoblasts (MB) and myotubes (MT) in differentiation medium (DM) for 72 hours. Venn diagram represents the number of proteins that bind mDia1 in MB or MT or both MB and MT. ( f ) Phb2 peptides identified in MT lysates by LC-MS/MS analysis of mDia1 IP proteins. Phb2 aa sequence (NCBI Reference Sequence # NP_031557.2) showing peptides identified in first (red), second and third (blue) and all three (underlined) biological replicates. ( g , h ) Reciprocal IP of endogenous mDia1 and Phb2 to identify stage-specific interaction. Lysates from proliferating MB (GM), MT in DM for 24 (D24) and 72 (D72) hours were harvested and subjected to IP with anti-mDia1 ( g ) or anti-Phb2 ( h ) antibodies. IP samples were loaded on different gels, blots were cut and processed in parallel, using same conditions of antibody incubation and exposure time during developing. ( i ) Western blot showing the expression profile of mDia1 and Phb2 in GM, D24, and D72 lysates. All the lysates were run on a single gel, blots were cut and probed for mDia1, Phb2, Akt2, MyoD, β-actin and GAPDH. The same lysates were run on a different gel, blots were cut and probed for MyoG, Akt1 and GAPDH. ( j , k ) Bar diagram represents the densitometric quantification of western blots shown in (i) *p

    Techniques Used: Mutagenesis, Binding Assay, Two Hybrid Screening, Transformation Assay, Positive Control, Negative Control, Activation Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, Liquid Chromatography with Mass Spectroscopy, Sequencing, Incubation, Expressing

    Co-expression of mDia1ΔN3 and Phb2 rescues MyoG promoter activity. C2C12 were transfected with various mDia1 and Phb2 mutants along with MyoG-promoter reporter construct and shifted to DM for 72 hours, followed by lysis and dual-luciferase assays. ( a ) Normalised MyoG promoter activity in MT transfected with mDia1ΔN3, mDia1H + P or Phb2 FL. *p
    Figure Legend Snippet: Co-expression of mDia1ΔN3 and Phb2 rescues MyoG promoter activity. C2C12 were transfected with various mDia1 and Phb2 mutants along with MyoG-promoter reporter construct and shifted to DM for 72 hours, followed by lysis and dual-luciferase assays. ( a ) Normalised MyoG promoter activity in MT transfected with mDia1ΔN3, mDia1H + P or Phb2 FL. *p

    Techniques Used: Expressing, Activity Assay, Transfection, Construct, Lysis, Luciferase

    23) Product Images from "Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia"

    Article Title: Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006079

    Expression and purification of RABV NP and characterization of antiserum. RABV NP inclusion bodies (IBs) (A) and gel-purified RABV NP (B) were resolved by SDS PAGE and stained with Coomassie blue. All lanes of gels stained with Coomassie blue were loaded with 10 μl of RABV NP in the dilutions or amounts indicated. Recombinant, gel-eluted His-tagged RABV NP was identified by immunoblotting with anti-His antibody (1:1,000) followed by sheep anti-mouse-HRP (1:2,000) (C). Sera from a pre- and post-immunized rabbit were diluted 1:10,000 and assessed for anti-RABV NP polyclonal antibody production by immunoblotting (D). All gels used for immunoblotting were loaded with 10 ng of RABV NP per well. Molecular mass markers were Mark 12 or See Blue Plus 2 (Invitrogen).
    Figure Legend Snippet: Expression and purification of RABV NP and characterization of antiserum. RABV NP inclusion bodies (IBs) (A) and gel-purified RABV NP (B) were resolved by SDS PAGE and stained with Coomassie blue. All lanes of gels stained with Coomassie blue were loaded with 10 μl of RABV NP in the dilutions or amounts indicated. Recombinant, gel-eluted His-tagged RABV NP was identified by immunoblotting with anti-His antibody (1:1,000) followed by sheep anti-mouse-HRP (1:2,000) (C). Sera from a pre- and post-immunized rabbit were diluted 1:10,000 and assessed for anti-RABV NP polyclonal antibody production by immunoblotting (D). All gels used for immunoblotting were loaded with 10 ng of RABV NP per well. Molecular mass markers were Mark 12 or See Blue Plus 2 (Invitrogen).

    Techniques Used: Expressing, Purification, SDS Page, Staining, Recombinant

    24) Product Images from "Methionine Residues in Exoproteins and Their Recycling by Methionine Sulfoxide Reductase AB Serve as an Antioxidant Strategy in Bacillus cereus"

    Article Title: Methionine Residues in Exoproteins and Their Recycling by Methionine Sulfoxide Reductase AB Serve as an Antioxidant Strategy in Bacillus cereus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.01342

    Altered growth and long-term survival of Δ msrAB mutant cells and complemented Δ msrAB /pHT304 msrAB cells. (A) Growth curves of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells in pH-regulated batch cultures under aerobiosis. (B) Long-term survival of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells after growth under aerobiosis. (C) Glucose consumption of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells. (D) Acetate production of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells. Significant differences are indicated with one ( p
    Figure Legend Snippet: Altered growth and long-term survival of Δ msrAB mutant cells and complemented Δ msrAB /pHT304 msrAB cells. (A) Growth curves of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells in pH-regulated batch cultures under aerobiosis. (B) Long-term survival of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells after growth under aerobiosis. (C) Glucose consumption of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells. (D) Acetate production of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells. Significant differences are indicated with one ( p

    Techniques Used: Mutagenesis

    Dynamics of Met(O) content in Δ msrAB mutant cells and complemented Δ msrAB /pHT304 msrAB cells. WT (blue), Δ msrAB (red), and Δ msrAB /pHT304msrAB (green) were grown in MOD medium as described in Figure 2 . The cells were harvested at EE, LE and S growth phases. The Met(O) content of the intracellular proteome (A) and exoproteome (B) was calculated as the percentage of the number of Met(O) vs. the total number of Met residues. Data are the means of triplicate measures obtained from three independent cultures at the EE, LE, and S growth phases. Significant differences between two strains are indicated with one ( p
    Figure Legend Snippet: Dynamics of Met(O) content in Δ msrAB mutant cells and complemented Δ msrAB /pHT304 msrAB cells. WT (blue), Δ msrAB (red), and Δ msrAB /pHT304msrAB (green) were grown in MOD medium as described in Figure 2 . The cells were harvested at EE, LE and S growth phases. The Met(O) content of the intracellular proteome (A) and exoproteome (B) was calculated as the percentage of the number of Met(O) vs. the total number of Met residues. Data are the means of triplicate measures obtained from three independent cultures at the EE, LE, and S growth phases. Significant differences between two strains are indicated with one ( p

    Techniques Used: Mutagenesis

    Growth phase-dependent changes of msrAB transcript levels in wild-type (blue) and complemented Δ msrAB /pHT304 msrAB (green) strains. Fold changes refer to the levels observed in early exponential (EE) phase cultures of the WT strains. Significant differences are indicated with one ( p
    Figure Legend Snippet: Growth phase-dependent changes of msrAB transcript levels in wild-type (blue) and complemented Δ msrAB /pHT304 msrAB (green) strains. Fold changes refer to the levels observed in early exponential (EE) phase cultures of the WT strains. Significant differences are indicated with one ( p

    Techniques Used:

    Exoproteome concentration and protease assay. Concentrations (A) and casein proteolytic activity (B) are indicated for total extracellular proteins of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells. Error bars represent the standard deviation from two independent measures for each biological triplicate. Significant differences ( p
    Figure Legend Snippet: Exoproteome concentration and protease assay. Concentrations (A) and casein proteolytic activity (B) are indicated for total extracellular proteins of WT (blue), Δ msrAB (red) and Δ msrAB /pHT304 msrAB (green) cells. Error bars represent the standard deviation from two independent measures for each biological triplicate. Significant differences ( p

    Techniques Used: Concentration Assay, Protease Assay, Activity Assay, Standard Deviation

    25) Product Images from "Abrin Toxicity and Bioavailability after Temperature and pH Treatment"

    Article Title: Abrin Toxicity and Bioavailability after Temperature and pH Treatment

    Journal: Toxins

    doi: 10.3390/toxins9100320

    Temperature effects on abrin toxicity in the intravenous mouse bioassay. ( A ) A series of known abrin samples were injected into mice tail vein injection (iv) to derive time-to-death standard curves and LD 50 values. Data was combined from two independent experiments from seven dosage levels consisting of a total of n = 6 to 8 mice per dose. The data was plotted using the log (inhibitor) vs. response (three parameter) curve on GraphPad Prism 6. R 2 = 0.8257; ( B ) Temperature treated toxins were administered to mice iv at a lethal dose of 1 μg per mouse ( n = 4 mice per experimental condition). Mice given abrin treated at 74 °C or higher all survived as compared to the untreated controls (**, p = 0.0082). The time-to-death delay seen from mice given the abrin treated at 63 °C was not statistically significant ( p = 0.1580). Two independent experiments were performed and one representative set of survival curves is shown. Survival curves were plotted for each condition and the log-rank (Mantel–Cox) test was used to evaluate statistical significance on GraphPad Prism 6.
    Figure Legend Snippet: Temperature effects on abrin toxicity in the intravenous mouse bioassay. ( A ) A series of known abrin samples were injected into mice tail vein injection (iv) to derive time-to-death standard curves and LD 50 values. Data was combined from two independent experiments from seven dosage levels consisting of a total of n = 6 to 8 mice per dose. The data was plotted using the log (inhibitor) vs. response (three parameter) curve on GraphPad Prism 6. R 2 = 0.8257; ( B ) Temperature treated toxins were administered to mice iv at a lethal dose of 1 μg per mouse ( n = 4 mice per experimental condition). Mice given abrin treated at 74 °C or higher all survived as compared to the untreated controls (**, p = 0.0082). The time-to-death delay seen from mice given the abrin treated at 63 °C was not statistically significant ( p = 0.1580). Two independent experiments were performed and one representative set of survival curves is shown. Survival curves were plotted for each condition and the log-rank (Mantel–Cox) test was used to evaluate statistical significance on GraphPad Prism 6.

    Techniques Used: Injection, Mouse Assay

    pH treatment of abrin has no detrimental effect on toxin activity. ( A ) A single concentration of 100 ng/mL abrin was used in cell free translation assays for all pH conditions. There is no significant difference between the various pH-exposed toxins. Values represent means of triplicate samples ± SD. Statistical significance was determined by two-tailed unpaired Student’s t -test. The data represents a single experiment; ( B ) pH-treated toxins were administered to mice iv at a lethal dose of 1 μg per mouse ( n = 5 mice per experimental condition) for one experiment. There was no detrimental effect on abrin’s ability to cause intoxication and subsequent death. Survival curves were plotted for mice for each condition and the log-rank (Mantel–Cox) test was used to evaluate statistical significance on GraphPad Prism 6. The only statistical significant decrease in toxicity was seen in abrin treated at pH 3.0 which shortened the time-to-death compared to abrin treated at pH 7.0 (** p = 0.0027).
    Figure Legend Snippet: pH treatment of abrin has no detrimental effect on toxin activity. ( A ) A single concentration of 100 ng/mL abrin was used in cell free translation assays for all pH conditions. There is no significant difference between the various pH-exposed toxins. Values represent means of triplicate samples ± SD. Statistical significance was determined by two-tailed unpaired Student’s t -test. The data represents a single experiment; ( B ) pH-treated toxins were administered to mice iv at a lethal dose of 1 μg per mouse ( n = 5 mice per experimental condition) for one experiment. There was no detrimental effect on abrin’s ability to cause intoxication and subsequent death. Survival curves were plotted for mice for each condition and the log-rank (Mantel–Cox) test was used to evaluate statistical significance on GraphPad Prism 6. The only statistical significant decrease in toxicity was seen in abrin treated at pH 3.0 which shortened the time-to-death compared to abrin treated at pH 7.0 (** p = 0.0027).

    Techniques Used: Activity Assay, Concentration Assay, Two Tailed Test, Mouse Assay

    SDS-PAGE analysis of heterogeneous toxin complexes and subunits. An amount of 100 ng per lane of sample (abrin, abrin A-chain, abrin B-chain) treated or not treated with 0.05 M DTT was loaded onto a NuPAGE 4–12% Bis-Tris gel and subjected to SDS-PAGE electrophoresis. The gel was silver stained with the SilverXpress kit. In the absence of the reducing agent, abrin predominantly consists of the holotoxin with small amounts of agglutinin, A-chain, and B-chain. Once reduced with DTT, abrin is almost all reduced to the smaller individual A- and B-chains. The abrin A-chain control sample with DTT has two predominant A-chain species. The reduced abrin B-chain control has one predominant species that is of higher molecular weight than the individual A-chain control.
    Figure Legend Snippet: SDS-PAGE analysis of heterogeneous toxin complexes and subunits. An amount of 100 ng per lane of sample (abrin, abrin A-chain, abrin B-chain) treated or not treated with 0.05 M DTT was loaded onto a NuPAGE 4–12% Bis-Tris gel and subjected to SDS-PAGE electrophoresis. The gel was silver stained with the SilverXpress kit. In the absence of the reducing agent, abrin predominantly consists of the holotoxin with small amounts of agglutinin, A-chain, and B-chain. Once reduced with DTT, abrin is almost all reduced to the smaller individual A- and B-chains. The abrin A-chain control sample with DTT has two predominant A-chain species. The reduced abrin B-chain control has one predominant species that is of higher molecular weight than the individual A-chain control.

    Techniques Used: SDS Page, Electrophoresis, Staining, Molecular Weight

    Translation inhibition by the plant toxin Abrin was abrogated by exposure to high temperature. ( A ) A representative standard curve shows increasing translation inhibition until saturation as one increased the abrin concentration in the in vitro cell free translation (CFT) assay run in parallel with the experimental samples shown in ( B ). Values represent means of triplicate samples ± SD. ( B ) Cell free translation assay using a single concentration of toxin (100 ng/mL) for all the different conditions. Increasing the temperature that abrin is exposed to decreases the translational inhibition seen in the CFT assay. Values represent means of triplicate samples ± SD. Statistical significance was determined by two-tailed unpaired Student’s t -test, (****) p
    Figure Legend Snippet: Translation inhibition by the plant toxin Abrin was abrogated by exposure to high temperature. ( A ) A representative standard curve shows increasing translation inhibition until saturation as one increased the abrin concentration in the in vitro cell free translation (CFT) assay run in parallel with the experimental samples shown in ( B ). Values represent means of triplicate samples ± SD. ( B ) Cell free translation assay using a single concentration of toxin (100 ng/mL) for all the different conditions. Increasing the temperature that abrin is exposed to decreases the translational inhibition seen in the CFT assay. Values represent means of triplicate samples ± SD. Statistical significance was determined by two-tailed unpaired Student’s t -test, (****) p

    Techniques Used: Inhibition, Concentration Assay, In Vitro, Two Tailed Test

    26) Product Images from "Direct competition between DNA binding factors highlights the role of Krüppel-like Factor 1 in the erythroid/megakaryocyte switch"

    Article Title: Direct competition between DNA binding factors highlights the role of Krüppel-like Factor 1 in the erythroid/megakaryocyte switch

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03289-5

    The GST-KLF1 DBD protein displaces endogenous KLF1 at a number of KLF1 target genes in vivo . ( A ) Schematic depicting the experimental design of the ChIP experiment. ( B ) ChIPs were performed using α-KLF1, α-GST and α-IgG in both in MEL + GST and MEL + GST-KLF1 DBD clones, (n = 4 for each group per IP). Data are represented as the fold-change enrichment of IP over input, where the negative control for each IP, the Klf1 promoter for GST, and the Klf8 +33 kb locus for both KLF1 and IgG, was set to 1. The Klf3 1b and Klf8 promoters have been included as positive controls while the Klf1 promoter, and Klf3 +10 kb and Klf8 +33 kb genomic regions are shown as negative controls. Error bars shown represent standard error of the mean, p values indicate the difference between the means, *p
    Figure Legend Snippet: The GST-KLF1 DBD protein displaces endogenous KLF1 at a number of KLF1 target genes in vivo . ( A ) Schematic depicting the experimental design of the ChIP experiment. ( B ) ChIPs were performed using α-KLF1, α-GST and α-IgG in both in MEL + GST and MEL + GST-KLF1 DBD clones, (n = 4 for each group per IP). Data are represented as the fold-change enrichment of IP over input, where the negative control for each IP, the Klf1 promoter for GST, and the Klf8 +33 kb locus for both KLF1 and IgG, was set to 1. The Klf3 1b and Klf8 promoters have been included as positive controls while the Klf1 promoter, and Klf3 +10 kb and Klf8 +33 kb genomic regions are shown as negative controls. Error bars shown represent standard error of the mean, p values indicate the difference between the means, *p

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Clone Assay, Negative Control

    The GST-KLF1 DBD construct is capable of binding to the canonical KLF1 binding site and has a biological effect in MELs. ( A ) Schematic demonstrating the experimental design of the two different constructs nucleofected into MEL cells. ( B ) Western blots from MEL lines showing protein expression of GST and GST-KLF1 DBD for each clone. Actin is presented as a loading control. ( C ) EMSAs from representative MEL clones indicating GST-KLF1 DBD is capable of binding the canonical KLF1 consensus sequence ( β - globin CACCC probe) in vitro . ( D ) Western blots from MEL clones with an antibody that recognises both endogenous KLF1 and GST-KLF1 DBD. β-Actin is presented as a loading control. ( E ) Western blots from MEL clones with an antibody that recognises KLF3, indicating reduced expression of endogenous KLF3 in cells overexpressing GST-KLF1 DBD compared to GST only clones. β-Actin is presented as a loading control. n = 4 for each construct.
    Figure Legend Snippet: The GST-KLF1 DBD construct is capable of binding to the canonical KLF1 binding site and has a biological effect in MELs. ( A ) Schematic demonstrating the experimental design of the two different constructs nucleofected into MEL cells. ( B ) Western blots from MEL lines showing protein expression of GST and GST-KLF1 DBD for each clone. Actin is presented as a loading control. ( C ) EMSAs from representative MEL clones indicating GST-KLF1 DBD is capable of binding the canonical KLF1 consensus sequence ( β - globin CACCC probe) in vitro . ( D ) Western blots from MEL clones with an antibody that recognises both endogenous KLF1 and GST-KLF1 DBD. β-Actin is presented as a loading control. ( E ) Western blots from MEL clones with an antibody that recognises KLF3, indicating reduced expression of endogenous KLF3 in cells overexpressing GST-KLF1 DBD compared to GST only clones. β-Actin is presented as a loading control. n = 4 for each construct.

    Techniques Used: Construct, Binding Assay, Western Blot, Expressing, Clone Assay, Sequencing, In Vitro

    GST-KLF1 DBD can act as a dominant negative at some KLF1 target sites to displace endogenous KLF1 binding, thereby inhibiting DNA activation. ( A ) In the absence of GST-KLF1 DBD, endogenous KLF1 is able to bind and activate its target genes normally. ( B ) At some KLF1 target sites, for instance the Klf3 1b promoter, the GST-KLF1 DBD protein is able to out-compete endogenous KLF1, either by sheer amount of protein present, or a higher affinity for the site. This results in reduced activation of these target genes. ( C ) At some sites, GST is unable to bind due to it not being able to interact efficiently with other KLF1 partners and SRC family members. In this case, activation of KLF1 target genes in unaffected.
    Figure Legend Snippet: GST-KLF1 DBD can act as a dominant negative at some KLF1 target sites to displace endogenous KLF1 binding, thereby inhibiting DNA activation. ( A ) In the absence of GST-KLF1 DBD, endogenous KLF1 is able to bind and activate its target genes normally. ( B ) At some KLF1 target sites, for instance the Klf3 1b promoter, the GST-KLF1 DBD protein is able to out-compete endogenous KLF1, either by sheer amount of protein present, or a higher affinity for the site. This results in reduced activation of these target genes. ( C ) At some sites, GST is unable to bind due to it not being able to interact efficiently with other KLF1 partners and SRC family members. In this case, activation of KLF1 target genes in unaffected.

    Techniques Used: Activated Clotting Time Assay, Dominant Negative Mutation, Binding Assay, Activation Assay

    27) Product Images from "A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization"

    Article Title: A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00219

    HP0248 is enriched in the DRM fractions of H. pylori cell membranes. (A) Whole cell lysates of H. pylori 251 WT, Δ FLOT , or FLOT ( FLOT +) bacteria were analyzed by Western blotting. Full length HP0248 protein (molecular weight, c . 40 kDa) was detected in WT and FLOT ( FLOT +) preparations, but not in those of the Δ FLOT strain. A non-specific protein band was also present in all three preparations. (B) Western Blot of H. pylori 251 WT whole cell lysate (lane 1), inner membrane (lane 2), outer membrane (lane 3) and cytoplasmic (lane 4) fractions. UreA was used as a loading control. (C) Coomassie-stained SDS-PAGE gel showing the total protein profiles of DRM (lane 1) and DSM (lane 2) preparations of H. pylori 251 WT. Molecular weight markers are shown. (D) Western blot of DRM (lane 1) and DSM (lane 2) preparations from H. pylori 251 WT, Δ FLOT , or FLOT ( FLOT +) bacteria. (E) Densitometric analysis of HP0248 in DRM and DSM fractions from H. pylori 251 WT, Δ FLOT , and FLOT ( FLOT +) bacteria. The relative amount of HP0248 in each fraction (mean ± SEM from three independent experiments) is expressed relative to that in the WT DRM fraction. Data were analyzed using the one-way ANOVA. ** P
    Figure Legend Snippet: HP0248 is enriched in the DRM fractions of H. pylori cell membranes. (A) Whole cell lysates of H. pylori 251 WT, Δ FLOT , or FLOT ( FLOT +) bacteria were analyzed by Western blotting. Full length HP0248 protein (molecular weight, c . 40 kDa) was detected in WT and FLOT ( FLOT +) preparations, but not in those of the Δ FLOT strain. A non-specific protein band was also present in all three preparations. (B) Western Blot of H. pylori 251 WT whole cell lysate (lane 1), inner membrane (lane 2), outer membrane (lane 3) and cytoplasmic (lane 4) fractions. UreA was used as a loading control. (C) Coomassie-stained SDS-PAGE gel showing the total protein profiles of DRM (lane 1) and DSM (lane 2) preparations of H. pylori 251 WT. Molecular weight markers are shown. (D) Western blot of DRM (lane 1) and DSM (lane 2) preparations from H. pylori 251 WT, Δ FLOT , or FLOT ( FLOT +) bacteria. (E) Densitometric analysis of HP0248 in DRM and DSM fractions from H. pylori 251 WT, Δ FLOT , and FLOT ( FLOT +) bacteria. The relative amount of HP0248 in each fraction (mean ± SEM from three independent experiments) is expressed relative to that in the WT DRM fraction. Data were analyzed using the one-way ANOVA. ** P

    Techniques Used: Western Blot, Molecular Weight, Staining, SDS Page

    28) Product Images from "Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice"

    Article Title: Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice

    Journal: Virology Journal

    doi: 10.1186/s12985-017-0789-2

    Immunofluorescence assay (IFA) and Western blot analysis for identification of G and N proteins expressed from recombinant adenoviruses. The IFA was performed in mock-infected 293A cells ( a ) and cells infected with recombinant adenoviruses Ad-0910 N ( b ), Ad-0910G ( c ), and Ad-0910Gsped ( d ). Cells were fixed 2 days post-infection and subjected to antibody staining using RABV G and N protein-specific antibodies. Specific fluorescence ( arrows ) was detected in 293A cells infected with each recombinant adenovirus. Bars , 100 μm. Western blot analysis was conducted using 293A cell lysates infected with Ad-0910G, Ad-0910Gsped and Ad-0910 N, and monoclonal antibodies against the G or N protein of RABV. ( e ) Lane 1, 293A cell lysates infected with Ad-0910G; lane 2, 293A cell lysates infected with Ad-0910Gsped; lanes 3 and 4, NG108–15 cell lysates infected with the ERA strain; lane 5, mock-infected 293A cell lysates, ( f ) lane 1, 293A cell lysates infected with Ad-0910 N; lane 2 NG108–15 cell lysates infected with the ERA strain; lane 3, mock-infected 293A cell lysates
    Figure Legend Snippet: Immunofluorescence assay (IFA) and Western blot analysis for identification of G and N proteins expressed from recombinant adenoviruses. The IFA was performed in mock-infected 293A cells ( a ) and cells infected with recombinant adenoviruses Ad-0910 N ( b ), Ad-0910G ( c ), and Ad-0910Gsped ( d ). Cells were fixed 2 days post-infection and subjected to antibody staining using RABV G and N protein-specific antibodies. Specific fluorescence ( arrows ) was detected in 293A cells infected with each recombinant adenovirus. Bars , 100 μm. Western blot analysis was conducted using 293A cell lysates infected with Ad-0910G, Ad-0910Gsped and Ad-0910 N, and monoclonal antibodies against the G or N protein of RABV. ( e ) Lane 1, 293A cell lysates infected with Ad-0910G; lane 2, 293A cell lysates infected with Ad-0910Gsped; lanes 3 and 4, NG108–15 cell lysates infected with the ERA strain; lane 5, mock-infected 293A cell lysates, ( f ) lane 1, 293A cell lysates infected with Ad-0910 N; lane 2 NG108–15 cell lysates infected with the ERA strain; lane 3, mock-infected 293A cell lysates

    Techniques Used: Immunofluorescence, Western Blot, Recombinant, Infection, Staining, Fluorescence

    Survival rates of mice immunized with recombinant adenoviruses expressing RABV G and N protein after RABV CVS-N2c challenge. Mice in each group were inoculated with 0.2 mL of each virus (Ad-0910G and Ad-0910Gsped) or mixed virus (Ad-0910G + Ad-0910 N and Ad-0910Gsped + Ad-0910 N) by the intramuscular route. Mice in the control group were inoculated with an equal volume of PBS. Five immunized mice from each group were challenged intramuscularly (IM) with 30 μL of CVS-N2c (25 LD 50 /0.03 mL) 21 days following inoculation of recombinant adenoviruses ( a ). The remaining five mice in each group were challenged via the intracranial (IC) route ( b ). After challenge, the survival of mice was checked daily for 3 weeks
    Figure Legend Snippet: Survival rates of mice immunized with recombinant adenoviruses expressing RABV G and N protein after RABV CVS-N2c challenge. Mice in each group were inoculated with 0.2 mL of each virus (Ad-0910G and Ad-0910Gsped) or mixed virus (Ad-0910G + Ad-0910 N and Ad-0910Gsped + Ad-0910 N) by the intramuscular route. Mice in the control group were inoculated with an equal volume of PBS. Five immunized mice from each group were challenged intramuscularly (IM) with 30 μL of CVS-N2c (25 LD 50 /0.03 mL) 21 days following inoculation of recombinant adenoviruses ( a ). The remaining five mice in each group were challenged via the intracranial (IC) route ( b ). After challenge, the survival of mice was checked daily for 3 weeks

    Techniques Used: Mouse Assay, Recombinant, Expressing

    Comparisons of amino acids sequences of G gene ( a ) and N gene ( b ) of Korean street rabies virus (KRVB0910) isolated from RABV-infected cattle with those of reference vaccine strain, ERA (AB781935). The dots indicate amino acids of transmembrane and cytoplasmic domains of G protein, which were removed for construction of Ad-0910Gsped. The asterisks (*) indicate different amino acids between KRVB0910 and ERA strain
    Figure Legend Snippet: Comparisons of amino acids sequences of G gene ( a ) and N gene ( b ) of Korean street rabies virus (KRVB0910) isolated from RABV-infected cattle with those of reference vaccine strain, ERA (AB781935). The dots indicate amino acids of transmembrane and cytoplasmic domains of G protein, which were removed for construction of Ad-0910Gsped. The asterisks (*) indicate different amino acids between KRVB0910 and ERA strain

    Techniques Used: Isolation, Infection

    29) Product Images from "Validation of Babesia proteasome as a drug target"

    Article Title: Validation of Babesia proteasome as a drug target

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    doi: 10.1016/j.ijpddr.2018.08.001

    Treatment of B. divergens ex vivo cultures: red blood cells infection. (A) Microscopy image of B. divergens ex vivo culture treated with 100 nM carfilzomib. (B) Microscopy image of B. divergens ex vivo culture treated with 0.008% DMSO. Smears were stained using DiffQuik staining set. Experimental conditions: starting parasitemia 2%, medium changed in 12 h intervals, total cultivation 48 h. Green dots indicate the parasitized red blood cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Treatment of B. divergens ex vivo cultures: red blood cells infection. (A) Microscopy image of B. divergens ex vivo culture treated with 100 nM carfilzomib. (B) Microscopy image of B. divergens ex vivo culture treated with 0.008% DMSO. Smears were stained using DiffQuik staining set. Experimental conditions: starting parasitemia 2%, medium changed in 12 h intervals, total cultivation 48 h. Green dots indicate the parasitized red blood cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Ex Vivo, Infection, Microscopy, Staining

    Treatment of B. divergens ex vivo cultures with peptide epoxyketone inhibitors. Structure and IC 50 determination of (A) carfilzomib, (B) epoxomicin, (C) ONX-0914. Data represent means of three independent biological replicates and the error bars indicate standard deviations. Smears were stained using DiffQuik staining set, parasitemia was counted at 1000 RBCs. Statistical analysis was performed in R using ANOVA (Kolmogorov-Smirnov test and the Bartlett test passed): * = p
    Figure Legend Snippet: Treatment of B. divergens ex vivo cultures with peptide epoxyketone inhibitors. Structure and IC 50 determination of (A) carfilzomib, (B) epoxomicin, (C) ONX-0914. Data represent means of three independent biological replicates and the error bars indicate standard deviations. Smears were stained using DiffQuik staining set, parasitemia was counted at 1000 RBCs. Statistical analysis was performed in R using ANOVA (Kolmogorov-Smirnov test and the Bartlett test passed): * = p

    Techniques Used: Ex Vivo, Staining

    Detection and inhibition of B. divergens proteasome β5 subunit activity. IC 50 values of all tested proteasome inhibitors on B. divergens crude cell lysates. Parasite lysates were obtained from ex vivo cultures and analysed using the fluorogenic peptidyl substrate, Suc-LLVY-AMC. Results represent the means of three independent replicates, error bars indicate standard deviations. IC 50 values were analysed using GraphPad Prism software (version 5).
    Figure Legend Snippet: Detection and inhibition of B. divergens proteasome β5 subunit activity. IC 50 values of all tested proteasome inhibitors on B. divergens crude cell lysates. Parasite lysates were obtained from ex vivo cultures and analysed using the fluorogenic peptidyl substrate, Suc-LLVY-AMC. Results represent the means of three independent replicates, error bars indicate standard deviations. IC 50 values were analysed using GraphPad Prism software (version 5).

    Techniques Used: Inhibition, Activity Assay, Ex Vivo, Software

    Treatment of B. divergens ex vivo cultures with peptide boronic acid inhibitors. Structure and IC 50 determination of (A) bortezomib and (B) ixazomib. Results represent means of three independent biological replicates, error bars indicate standard deviations. Smears were stained using DiffQuik staining set, parasitemia was counted at 1000 RBCs. Statistical analysis was performed in R using ANOVA (Kolmogorov Smirnov test and the Bartlett test passed): * = p
    Figure Legend Snippet: Treatment of B. divergens ex vivo cultures with peptide boronic acid inhibitors. Structure and IC 50 determination of (A) bortezomib and (B) ixazomib. Results represent means of three independent biological replicates, error bars indicate standard deviations. Smears were stained using DiffQuik staining set, parasitemia was counted at 1000 RBCs. Statistical analysis was performed in R using ANOVA (Kolmogorov Smirnov test and the Bartlett test passed): * = p

    Techniques Used: Ex Vivo, Staining

    30) Product Images from "CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa"

    Article Title: CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01757

    Structure of CL-LK and antibody specificity. (A) Schematic illustration of the domain organization of CL-L1 and CL-K1 polypeptide chains. The symbol “ * ”on the CL-L1 polypeptide chain represent two N-linked glycosylation sites in the carbohydrate recognition domain. CL-K1 is found in the circulation in the form of two isoforms: CL-K1a represents full-length and CL-K1d represents an alternative spliced form devoid of a part of the collagen-like region. (B) Subunit of CL-LK and oligomeric structures. A total of three polypeptide chains of CL-K1 and CL-L1 join to form a heteromeric subunit, which may further oligomerize into structures ranging from dimers to hexamers of subunits, here illustrated by a tetramer. (C) Analysis of purified CL-LK by SDS-PAGE and visualization by silver staining. The three bands of CL-L1 represent non-, partially, and fully glycosylated forms of CL-L1. (D,E) Specificity of monoclonal antibodies by Western blotting of serum under. reducing conditions and visualization by ECL. (F–H) SPR analyses of monoclonal antibodies with immobilized CL-K1, CL-L1, and CL-LK as antigen, respectively. MAbs 16-13 ( -- ) and 11-1 ( --- ) were analyzed for binding to immobilized purified CL-L1 (F) , CL-K1 (G) , or CL-KL (H) MAb using concentrations ranging from 0.1 to 10 µg/ml.
    Figure Legend Snippet: Structure of CL-LK and antibody specificity. (A) Schematic illustration of the domain organization of CL-L1 and CL-K1 polypeptide chains. The symbol “ * ”on the CL-L1 polypeptide chain represent two N-linked glycosylation sites in the carbohydrate recognition domain. CL-K1 is found in the circulation in the form of two isoforms: CL-K1a represents full-length and CL-K1d represents an alternative spliced form devoid of a part of the collagen-like region. (B) Subunit of CL-LK and oligomeric structures. A total of three polypeptide chains of CL-K1 and CL-L1 join to form a heteromeric subunit, which may further oligomerize into structures ranging from dimers to hexamers of subunits, here illustrated by a tetramer. (C) Analysis of purified CL-LK by SDS-PAGE and visualization by silver staining. The three bands of CL-L1 represent non-, partially, and fully glycosylated forms of CL-L1. (D,E) Specificity of monoclonal antibodies by Western blotting of serum under. reducing conditions and visualization by ECL. (F–H) SPR analyses of monoclonal antibodies with immobilized CL-K1, CL-L1, and CL-LK as antigen, respectively. MAbs 16-13 ( -- ) and 11-1 ( --- ) were analyzed for binding to immobilized purified CL-L1 (F) , CL-K1 (G) , or CL-KL (H) MAb using concentrations ranging from 0.1 to 10 µg/ml.

    Techniques Used: Purification, SDS Page, Silver Staining, Western Blot, SPR Assay, Binding Assay

    31) Product Images from "Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production"

    Article Title: Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197656

    Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) SDS PAGE of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).
    Figure Legend Snippet: Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) SDS PAGE of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).

    Techniques Used: Affinity Purification, Produced, Purification, Enzyme-linked Immunosorbent Assay, SDS Page, Polyacrylamide Gel Electrophoresis, SDS-Gel, Staining

    SDS-PAGE analysis of A244_N332 rgp120 HIV produced in 5F MGAT1 - CHO and CHO-S cells treated with PNGase or EndoH. Enzymes and buffers were purchased from (New England Biolabs, Ipswich, MA). Purified protein was denatured and reduced then incubated overnight at 37°C with or without glycosidase. Protein was resolved (2 μg/lane) on 4–12% SDS-PAGE gel and stained with Simply Blue. Plus (+) indicates enzyme treatment, minus indicates untreated.
    Figure Legend Snippet: SDS-PAGE analysis of A244_N332 rgp120 HIV produced in 5F MGAT1 - CHO and CHO-S cells treated with PNGase or EndoH. Enzymes and buffers were purchased from (New England Biolabs, Ipswich, MA). Purified protein was denatured and reduced then incubated overnight at 37°C with or without glycosidase. Protein was resolved (2 μg/lane) on 4–12% SDS-PAGE gel and stained with Simply Blue. Plus (+) indicates enzyme treatment, minus indicates untreated.

    Techniques Used: SDS Page, Produced, Purification, Incubation, Staining

    32) Product Images from "Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates"

    Article Title: Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12364-w

    IRES-mediated cell-free synthesis of different antibody constructs (IgG, scFv-Fc, scFv) using the CHO cell-free system. ( a ) Schematic representation of the antibody formats synthesized in this study. ( b ) Schematic representation of the DNA template design applied in this study. Antibody light chain (LC) contains one variable domain (V L ) and one constant domain (C L ). Antibody heavy chain (HC) is composed of one variable domain (V H ) and three constant domains (C H 1, C H 2, and C H 3). ( c ) Diagram showing protein yields determined in the complete translation mixture (TM), the supernatant fraction after centrifugation (SN1) and the microsomal fraction (MF). Protein synthesis reactions were performed in the presence of 14 C-leucine for subsequent qualitative and quantitative analysis. Standard deviations were calculated from triplicate analysis. ( d ) Qualitative analysis of cell-free synthesized proteins by SDS-PAGE and subsequent autoradiography. ( e ) Autoradiograph derived from SDS-PAGE gel showing glycosylation of HC in MF by digestion with EndoH and PNGase. scFv: single-chain variabe fragment; scFv-Fc: single-chain variable fragment Fc fusion.
    Figure Legend Snippet: IRES-mediated cell-free synthesis of different antibody constructs (IgG, scFv-Fc, scFv) using the CHO cell-free system. ( a ) Schematic representation of the antibody formats synthesized in this study. ( b ) Schematic representation of the DNA template design applied in this study. Antibody light chain (LC) contains one variable domain (V L ) and one constant domain (C L ). Antibody heavy chain (HC) is composed of one variable domain (V H ) and three constant domains (C H 1, C H 2, and C H 3). ( c ) Diagram showing protein yields determined in the complete translation mixture (TM), the supernatant fraction after centrifugation (SN1) and the microsomal fraction (MF). Protein synthesis reactions were performed in the presence of 14 C-leucine for subsequent qualitative and quantitative analysis. Standard deviations were calculated from triplicate analysis. ( d ) Qualitative analysis of cell-free synthesized proteins by SDS-PAGE and subsequent autoradiography. ( e ) Autoradiograph derived from SDS-PAGE gel showing glycosylation of HC in MF by digestion with EndoH and PNGase. scFv: single-chain variabe fragment; scFv-Fc: single-chain variable fragment Fc fusion.

    Techniques Used: Construct, Synthesized, Centrifugation, SDS Page, Autoradiography, Derivative Assay

    33) Product Images from "ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library"

    Article Title: ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174909

    Strategy for protein-analysis and vector validation (A) Schematic representation of the pEBB vector containing biotinylation target-domain (BTD) and (B) Simultaneous two-color target analysis of ANKRD54 wt and Δ3 mutant in Cos7 cells. The first three lanes (from left side of the blot) represent immunoprecipitation (IP) with streptavidin (SA) beads and the last three lanes show whole cell lysate (WCL). Anti-ANKRD54 (green) primary antibody recognizes the C-terminus of the protein and anti-IRDye 680-Streptavidin (red) recognizes the BTD domain.
    Figure Legend Snippet: Strategy for protein-analysis and vector validation (A) Schematic representation of the pEBB vector containing biotinylation target-domain (BTD) and (B) Simultaneous two-color target analysis of ANKRD54 wt and Δ3 mutant in Cos7 cells. The first three lanes (from left side of the blot) represent immunoprecipitation (IP) with streptavidin (SA) beads and the last three lanes show whole cell lysate (WCL). Anti-ANKRD54 (green) primary antibody recognizes the C-terminus of the protein and anti-IRDye 680-Streptavidin (red) recognizes the BTD domain.

    Techniques Used: Plasmid Preparation, Mutagenesis, Immunoprecipitation

    34) Product Images from "The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human"

    Article Title: The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20180248

    Comparing LRRK2-mediated Rab10 phosphorylation in neutrophils from control, idiopathic, and VPS35[D620N] Parkinson's patients. Neutrophils were isolated from nine age-matched idiopathic PD patients, nine non-age-matched healthy controls, and three individuals with a heterozygous VPS35[D620N] mutation with PD. Demographic and clinical data for each subject analysed are provided in Supplementary Table S1. Cells were treated with or without 200 nM MLi-2 for 30 min. Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml primary antibody concentration), and the membranes developed using the Odyssey CLx scan Western Blot imaging system. ( A ) Samples from nine age-matched idiopathic PD patients controls and three individuals with a heterozygous VPS35[D620N] mutation with PD were analysed (left and middle panel) by immunoblotting analysis with the indicated antibodies. Samples analysed on the left hand and middle panels were generated ∼8 weeks apart and analysed on different gels. The same internal standard was run on every gel to compare the different sets of samples. Similar results were obtained in two independent immunoblot experiments of the same extracts. Immunoblots from were quantified for phospho-Thr73 Rab10/total Rab10 ratio (right panel). Data were analysed by one-way ANOVA with Tukey's multiple comparison test. Data presented as means ± SD; *** P
    Figure Legend Snippet: Comparing LRRK2-mediated Rab10 phosphorylation in neutrophils from control, idiopathic, and VPS35[D620N] Parkinson's patients. Neutrophils were isolated from nine age-matched idiopathic PD patients, nine non-age-matched healthy controls, and three individuals with a heterozygous VPS35[D620N] mutation with PD. Demographic and clinical data for each subject analysed are provided in Supplementary Table S1. Cells were treated with or without 200 nM MLi-2 for 30 min. Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml primary antibody concentration), and the membranes developed using the Odyssey CLx scan Western Blot imaging system. ( A ) Samples from nine age-matched idiopathic PD patients controls and three individuals with a heterozygous VPS35[D620N] mutation with PD were analysed (left and middle panel) by immunoblotting analysis with the indicated antibodies. Samples analysed on the left hand and middle panels were generated ∼8 weeks apart and analysed on different gels. The same internal standard was run on every gel to compare the different sets of samples. Similar results were obtained in two independent immunoblot experiments of the same extracts. Immunoblots from were quantified for phospho-Thr73 Rab10/total Rab10 ratio (right panel). Data were analysed by one-way ANOVA with Tukey's multiple comparison test. Data presented as means ± SD; *** P

    Techniques Used: Isolation, Mutagenesis, Concentration Assay, Western Blot, Imaging, Generated

    Comparing LRRK2-mediated Rab10 phosphorylation in monocytes from control, idiopathic, and VPS35[D620N] Parkinson's patients. Monocytes were isolated from nine age-matched idiopathic PD patients, nine non-age-matched healthy controls, and three individuals with a heterozygous VPS35[D620N] mutation with PD. Demographic and clinical data for each subject analysed are provided in Supplementary Table S1. Cells were treated with or without 200 nM MLi-2 for 30 min. Monocytes were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml primary antibody concentration), and the membranes developed using the Odyssey CLx scan Western Blot imaging system. ( A ) Samples from nine age-matched idiopathic PD patients controls, and three individuals with a heterozygous VPS35[D620N] mutation with PD were analysed (left and middle panel) by immunoblotting analysis with the indicated antibodies. Samples analysed on the left hand and middle panels were generated ∼8 weeks apart and analysed on different gels with immunoblotting undertaken in parallel. The same internal standard was run on every gel to compare samples run on different gels. Similar results were obtained in two independent immunoblot experiments of the same extracts. Immunoblots from were quantified for phospho-Thr73 Rab10/total Rab10 ratio (right panel). Data were analysed by one-way ANOVA with Tukey's multiple comparison test. Data presented as means ± SD; *** P
    Figure Legend Snippet: Comparing LRRK2-mediated Rab10 phosphorylation in monocytes from control, idiopathic, and VPS35[D620N] Parkinson's patients. Monocytes were isolated from nine age-matched idiopathic PD patients, nine non-age-matched healthy controls, and three individuals with a heterozygous VPS35[D620N] mutation with PD. Demographic and clinical data for each subject analysed are provided in Supplementary Table S1. Cells were treated with or without 200 nM MLi-2 for 30 min. Monocytes were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml primary antibody concentration), and the membranes developed using the Odyssey CLx scan Western Blot imaging system. ( A ) Samples from nine age-matched idiopathic PD patients controls, and three individuals with a heterozygous VPS35[D620N] mutation with PD were analysed (left and middle panel) by immunoblotting analysis with the indicated antibodies. Samples analysed on the left hand and middle panels were generated ∼8 weeks apart and analysed on different gels with immunoblotting undertaken in parallel. The same internal standard was run on every gel to compare samples run on different gels. Similar results were obtained in two independent immunoblot experiments of the same extracts. Immunoblots from were quantified for phospho-Thr73 Rab10/total Rab10 ratio (right panel). Data were analysed by one-way ANOVA with Tukey's multiple comparison test. Data presented as means ± SD; *** P

    Techniques Used: Isolation, Mutagenesis, Concentration Assay, Western Blot, Imaging, Generated

    35) Product Images from "Evaluation of glycoprotein Ov8 as a potential antigen for an OvHV-2-specific diagnostic assay"

    Article Title: Evaluation of glycoprotein Ov8 as a potential antigen for an OvHV-2-specific diagnostic assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200130

    Ov8FC schematic representation and expression. (A) Schematic representation of full length Ov8 (Ov8), truncated Ov8 with Fc region of human IgG1 and His-tag (Ov8FC), and the Fc region of human IgG1 only with His-tag (hIgGFC, Negative control). Numbers below constructs indicate amino acids. (B) Western blot analysis of supernatant from 293-T cells transfected with pJP008-Ov8FC (Lane 1), pJP008 empty plasmid (Lane 2), protein markers (Lane 3) and pJP008-hIgGFC (Lane 4) using mouse monoclonal anti-His antibody. Molecular masses are indicated on the left in kilodalton (kDa). (C) Reverse transcription PCR of cells transfected with pJP008-Ov8FC with and without reverse transcriptase, respectively (Lanes 2 and 3) and pJP008 empty plasmid with and without reverse transcriptase, respectively (Lanes 4 and 5). Control PCR of pJP008-Ov8FC plasmid (Lane 6). DNA marker in kilo base pairs (Lane 1).
    Figure Legend Snippet: Ov8FC schematic representation and expression. (A) Schematic representation of full length Ov8 (Ov8), truncated Ov8 with Fc region of human IgG1 and His-tag (Ov8FC), and the Fc region of human IgG1 only with His-tag (hIgGFC, Negative control). Numbers below constructs indicate amino acids. (B) Western blot analysis of supernatant from 293-T cells transfected with pJP008-Ov8FC (Lane 1), pJP008 empty plasmid (Lane 2), protein markers (Lane 3) and pJP008-hIgGFC (Lane 4) using mouse monoclonal anti-His antibody. Molecular masses are indicated on the left in kilodalton (kDa). (C) Reverse transcription PCR of cells transfected with pJP008-Ov8FC with and without reverse transcriptase, respectively (Lanes 2 and 3) and pJP008 empty plasmid with and without reverse transcriptase, respectively (Lanes 4 and 5). Control PCR of pJP008-Ov8FC plasmid (Lane 6). DNA marker in kilo base pairs (Lane 1).

    Techniques Used: Expressing, Negative Control, Construct, Western Blot, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Marker

    36) Product Images from "Bacterial sepsis triggers an antiviral response that causes translation shutdown"

    Article Title: Bacterial sepsis triggers an antiviral response that causes translation shutdown

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI123284

    ISRIB reverses the effect of p-Eif2α on translation. ( A ) Ribo-Seq analysis of kidney extracts from mice treated with LPS for 16 hours with or without ISRIB. Smear plot in which top 10 differentially translated genes are highlighted in red (edgeR exactTest with BH adjusted P values
    Figure Legend Snippet: ISRIB reverses the effect of p-Eif2α on translation. ( A ) Ribo-Seq analysis of kidney extracts from mice treated with LPS for 16 hours with or without ISRIB. Smear plot in which top 10 differentially translated genes are highlighted in red (edgeR exactTest with BH adjusted P values

    Techniques Used: Mouse Assay

    Translation shutdown mediated by the Eif2ak2/Eif2α axis contributes to sepsis-induced kidney injury. ( A ) Mice were treated with 5 mg/kg LPS i.v. for indicated durations, and kidneys were analyzed by Western blot for Eif2ak2, Eif2α, and Ser51 p-Eif2α (arrow). ( B ) Select protein levels as determined by nascent proteomics are shown. ( C and D ) In vivo effects of ISRIB (5 mg/kg i.p.) on protein synthesis (puromycin incorporation in the kidney; ISRIB for 16 hours) and tissue expression of Eif2ak2, Eif2α, and p-Eif2α (kidneys harvested 16 hours after 5 mg/kg LPS i.v. with or without ISRIB i.p. for 16 hours). LPS increased Eif2ak2 and p-Eif2α. This increase was not affected by ISRIB. ( E ) Serum creatinine levels 24 hours after LPS with or without ISRIB treatment administered at indicated time points. * P
    Figure Legend Snippet: Translation shutdown mediated by the Eif2ak2/Eif2α axis contributes to sepsis-induced kidney injury. ( A ) Mice were treated with 5 mg/kg LPS i.v. for indicated durations, and kidneys were analyzed by Western blot for Eif2ak2, Eif2α, and Ser51 p-Eif2α (arrow). ( B ) Select protein levels as determined by nascent proteomics are shown. ( C and D ) In vivo effects of ISRIB (5 mg/kg i.p.) on protein synthesis (puromycin incorporation in the kidney; ISRIB for 16 hours) and tissue expression of Eif2ak2, Eif2α, and p-Eif2α (kidneys harvested 16 hours after 5 mg/kg LPS i.v. with or without ISRIB i.p. for 16 hours). LPS increased Eif2ak2 and p-Eif2α. This increase was not affected by ISRIB. ( E ) Serum creatinine levels 24 hours after LPS with or without ISRIB treatment administered at indicated time points. * P

    Techniques Used: Mouse Assay, Western Blot, In Vivo, Expressing

    37) Product Images from "Different Requirements for Scavenger Receptor Class B Type I in Hepatitis C Virus Cell-Free versus Cell-to-Cell Transmission"

    Article Title: Different Requirements for Scavenger Receptor Class B Type I in Hepatitis C Virus Cell-Free versus Cell-to-Cell Transmission

    Journal: Journal of Virology

    doi: 10.1128/JVI.01102-13

    Receptor requirements for cell-free infectivity with J6/JFH-1 and clone 2. (A, B) J6/JFH-1 or clone 2 cell-free infection efficiency in the presence of anti-CD81 (A) or anti-SR-BI (B) mAb. Huh-7.5 cells were preincubated with the indicated concentrations of mAb (μg/ml; log 10 ) and infected with HCVcc in the presence of the mAb. The percentage of HCV-infected cells at 48 hpi was measured by flow cytometry using Alexa Fluor 647-conjugated anti-NS5A antibody (9E10-AF647). MFIs obtained with isotype-matched controls were subtracted. Curves were extrapolated by fitting experimental data with GraphPad Prism (version 5.0) software. (C) Immunoblot for OCLN, CLDN1, and β-actin expression in Huh-7.5 cells expressing shIRR, shHCV, shOCLN, and shCLDN1. Cells were harvested at 72 h posttransduction, and 30 μg total cell lysate was loaded per lane. (D) Huh-7.5 cells expressing the indicated shRNAs were inoculated with J6/JFH-1 or clone 2 at an MOI of 0.1, and HCV infection frequency was measured at 48 hpi at the flow cytometer by staining the cells for NS5A with 9E10-AF647. The value for parental Huh-7.5 cells was set to 100%. Mean values and SDs from three independent experiments are plotted.
    Figure Legend Snippet: Receptor requirements for cell-free infectivity with J6/JFH-1 and clone 2. (A, B) J6/JFH-1 or clone 2 cell-free infection efficiency in the presence of anti-CD81 (A) or anti-SR-BI (B) mAb. Huh-7.5 cells were preincubated with the indicated concentrations of mAb (μg/ml; log 10 ) and infected with HCVcc in the presence of the mAb. The percentage of HCV-infected cells at 48 hpi was measured by flow cytometry using Alexa Fluor 647-conjugated anti-NS5A antibody (9E10-AF647). MFIs obtained with isotype-matched controls were subtracted. Curves were extrapolated by fitting experimental data with GraphPad Prism (version 5.0) software. (C) Immunoblot for OCLN, CLDN1, and β-actin expression in Huh-7.5 cells expressing shIRR, shHCV, shOCLN, and shCLDN1. Cells were harvested at 72 h posttransduction, and 30 μg total cell lysate was loaded per lane. (D) Huh-7.5 cells expressing the indicated shRNAs were inoculated with J6/JFH-1 or clone 2 at an MOI of 0.1, and HCV infection frequency was measured at 48 hpi at the flow cytometer by staining the cells for NS5A with 9E10-AF647. The value for parental Huh-7.5 cells was set to 100%. Mean values and SDs from three independent experiments are plotted.

    Techniques Used: Infection, Flow Cytometry, Cytometry, Software, Expressing, Staining

    38) Product Images from "5-Aminolevulinic acid induced apoptosis via oxidative stress in normal gastric epithelial cells"

    Article Title: 5-Aminolevulinic acid induced apoptosis via oxidative stress in normal gastric epithelial cells

    Journal: Journal of Clinical Biochemistry and Nutrition

    doi: 10.3164/jcbn.18-46

    Protein expression transitions after treatment with 50 µM 5-ALA for 48 h analysed by western blotting. (A) The expression level of cleaved caspase-9. 5-ALA enhanced expression in RGM cells. While the basal level was higher in RGK cells, 5-ALA did not markedly alter the level. (B) The level of phosphorylated p53 protein. Elevated level of phosphorylated p53 was observed in RGM cells after 5-ALA exposure. Expression was prone to decrease in RGK cells after 5-ALA treatment.
    Figure Legend Snippet: Protein expression transitions after treatment with 50 µM 5-ALA for 48 h analysed by western blotting. (A) The expression level of cleaved caspase-9. 5-ALA enhanced expression in RGM cells. While the basal level was higher in RGK cells, 5-ALA did not markedly alter the level. (B) The level of phosphorylated p53 protein. Elevated level of phosphorylated p53 was observed in RGM cells after 5-ALA exposure. Expression was prone to decrease in RGK cells after 5-ALA treatment.

    Techniques Used: Expressing, Western Blot

    39) Product Images from "Respiratory supercomplexes act as a platform for complex III‐mediated maturation of human mitochondrial complexes I and IV"

    Article Title: Respiratory supercomplexes act as a platform for complex III‐mediated maturation of human mitochondrial complexes I and IV

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102817

    Knock‐down of either GHITM or CHCHD3 does not produce cIII2 functional nor assembly defects (related to Fig 4 ) Oxygen consumption rates measured in WT cells transduced with lentiviral vectors encoding two different shRNAs specific for GHITM mRNA (shRNA GHITM 1 and shRNA GHITM 2) and with pLKO.1 without any shRNA insert (empty vector, EV). Respiration was measured in whole cells in the basal state (Routine), in the presence of oligomycin (Leak) and uncoupled with CCCP (ETS capacity), using a O2K high‐resolution respirometer (Oroboros instruments). The plotted values are the mean ± SD ( n = 4 for WT EV and WT shRNA GHITM 1 and n = 3 for WT shRNA GHITM 2). MRC enzyme activities normalized to the activity of citrate synthase (CS) measured in the same cell lines shown in (A). The plotted values are the mean ± SD ( n = 2 biological replicates). Two‐way ANOVA Tukey's multiple comparisons test * P = 0.0462 (CIV). SDS–PAGE, Western blot, and immunodetection analysis with the indicated specific antibodies. 1D BNGE, Western blot, and immunodetection analysis of digitonin‐solubilized samples from the same three cell lines shown in (A). Oxygen consumption rates measured in 143B WT cells transduced with lentiviral vectors encoding two different shRNAs specific for CHCHD3 mRNA (shRNA CHCHD3 1 and shRNA CHCHD3 2) and with pLKO.1 without any shRNA insert (empty vector, EV). Respiration was measured in whole cells in the basal state (Routine), in the presence of oligomycin (Leak) and uncoupled with CCCP (ETS capacity), using a O2K high‐resolution respirometer (Oroboros instruments). The plotted values are the mean ± SD ( n = 4). Two‐way ANOVA Tukey's multiple comparisons test * P = 0.0126 (Routine shRNA 2); * P = 0.0386 (ETS capacity shRNA 1); *** P = 0.0002 (ETS capacity shRNA 2). MRC enzyme activities normalized to the activity of citrate synthase (CS) measured in the same cell lines shown in (E). The plotted values are the mean ± SD ( n = 4 biological replicates). SDS–PAGE, Western blot, and immunodetection analysis with the indicated specific antibodies. 1D BNGE, Western blot, and immunodetection analysis of digitonin‐solubilized samples from the same three cell lines shown in (E). Source data are available online for this figure.
    Figure Legend Snippet: Knock‐down of either GHITM or CHCHD3 does not produce cIII2 functional nor assembly defects (related to Fig 4 ) Oxygen consumption rates measured in WT cells transduced with lentiviral vectors encoding two different shRNAs specific for GHITM mRNA (shRNA GHITM 1 and shRNA GHITM 2) and with pLKO.1 without any shRNA insert (empty vector, EV). Respiration was measured in whole cells in the basal state (Routine), in the presence of oligomycin (Leak) and uncoupled with CCCP (ETS capacity), using a O2K high‐resolution respirometer (Oroboros instruments). The plotted values are the mean ± SD ( n = 4 for WT EV and WT shRNA GHITM 1 and n = 3 for WT shRNA GHITM 2). MRC enzyme activities normalized to the activity of citrate synthase (CS) measured in the same cell lines shown in (A). The plotted values are the mean ± SD ( n = 2 biological replicates). Two‐way ANOVA Tukey's multiple comparisons test * P = 0.0462 (CIV). SDS–PAGE, Western blot, and immunodetection analysis with the indicated specific antibodies. 1D BNGE, Western blot, and immunodetection analysis of digitonin‐solubilized samples from the same three cell lines shown in (A). Oxygen consumption rates measured in 143B WT cells transduced with lentiviral vectors encoding two different shRNAs specific for CHCHD3 mRNA (shRNA CHCHD3 1 and shRNA CHCHD3 2) and with pLKO.1 without any shRNA insert (empty vector, EV). Respiration was measured in whole cells in the basal state (Routine), in the presence of oligomycin (Leak) and uncoupled with CCCP (ETS capacity), using a O2K high‐resolution respirometer (Oroboros instruments). The plotted values are the mean ± SD ( n = 4). Two‐way ANOVA Tukey's multiple comparisons test * P = 0.0126 (Routine shRNA 2); * P = 0.0386 (ETS capacity shRNA 1); *** P = 0.0002 (ETS capacity shRNA 2). MRC enzyme activities normalized to the activity of citrate synthase (CS) measured in the same cell lines shown in (E). The plotted values are the mean ± SD ( n = 4 biological replicates). SDS–PAGE, Western blot, and immunodetection analysis with the indicated specific antibodies. 1D BNGE, Western blot, and immunodetection analysis of digitonin‐solubilized samples from the same three cell lines shown in (E). Source data are available online for this figure.

    Techniques Used: Functional Assay, Transduction, shRNA, Plasmid Preparation, Activity Assay, SDS Page, Western Blot, Immunodetection

    Blue‐native gel electrophoresis (BNGE) mass spectrometry and immunodetection analysis of cIII 2 ‐related proteins Complexome profiles of cIII 2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised (see also Figs EV1 and EV2 ). The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, versus the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4‐CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Second‐dimension BNGE of digitonin‐solubilized samples from WT and Δ4‐CYB cells, Western blot and immunodetection of the indicated cIII 2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles. Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII 2 subunits. The x ‐axis values were the slice number (1‐64), and the y ‐axis values were the relative peptide intensity. The graph shows the mean ± SD ( n = 2). Two‐way ANOVA with Sidak's multiple comparisons test ** P = 0.0083 (UQCRC2); ** P = 0.0033 (UQCRFS1); * P = 0.0224; n.s. = non‐significant. Complexome profiles of two cIII 2 assembly factors (BCS1L and LYRM7 or MZM1L) generated in the same way as in (A). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Source data are available online for this figure.
    Figure Legend Snippet: Blue‐native gel electrophoresis (BNGE) mass spectrometry and immunodetection analysis of cIII 2 ‐related proteins Complexome profiles of cIII 2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised (see also Figs EV1 and EV2 ). The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, versus the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4‐CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Second‐dimension BNGE of digitonin‐solubilized samples from WT and Δ4‐CYB cells, Western blot and immunodetection of the indicated cIII 2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles. Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII 2 subunits. The x ‐axis values were the slice number (1‐64), and the y ‐axis values were the relative peptide intensity. The graph shows the mean ± SD ( n = 2). Two‐way ANOVA with Sidak's multiple comparisons test ** P = 0.0083 (UQCRC2); ** P = 0.0033 (UQCRFS1); * P = 0.0224; n.s. = non‐significant. Complexome profiles of two cIII 2 assembly factors (BCS1L and LYRM7 or MZM1L) generated in the same way as in (A). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Source data are available online for this figure.

    Techniques Used: Nucleic Acid Electrophoresis, Mass Spectrometry, Immunodetection, Generated, Labeling, Western Blot

    Assembly state of cV, cII, and cIV in Δ4‐CYB cells Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Fig 3 , but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al , 2018 ) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. Complexome profiles of the three detected cII subunits. The graphs were generated as in Fig 3 . The represented values are the mean ± SEM of the two reciprocal labeling experiments. Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). The graphs were generated as in Fig 3 , but in this case, the peptide intensity values for the individual subunits belonging to each assembly module (Vidoni et al , 2017 ) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve (calculated as in Fig 3 C) in the profiles corresponding to each cIV module. The plotted values are mean ± SD ( n = 2). Two‐way ANOVA with Sidak's multiple comparisons test ** P = 0.0088 (Early); ** P = 0.0063 (MT‐CO2); ** P = 0.0040 (NDUFA4); * P = 0.0264. 1D BNGE, Western blot, and immunodetection of two cIV subunits (MT‐CO1 and COX7B) in samples from WT and Δ4‐CYB cells solubilized with DDM and digitonin (Dig). Source data are available online for this figure.
    Figure Legend Snippet: Assembly state of cV, cII, and cIV in Δ4‐CYB cells Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Fig 3 , but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al , 2018 ) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. Complexome profiles of the three detected cII subunits. The graphs were generated as in Fig 3 . The represented values are the mean ± SEM of the two reciprocal labeling experiments. Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). The graphs were generated as in Fig 3 , but in this case, the peptide intensity values for the individual subunits belonging to each assembly module (Vidoni et al , 2017 ) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve (calculated as in Fig 3 C) in the profiles corresponding to each cIV module. The plotted values are mean ± SD ( n = 2). Two‐way ANOVA with Sidak's multiple comparisons test ** P = 0.0088 (Early); ** P = 0.0063 (MT‐CO2); ** P = 0.0040 (NDUFA4); * P = 0.0264. 1D BNGE, Western blot, and immunodetection of two cIV subunits (MT‐CO1 and COX7B) in samples from WT and Δ4‐CYB cells solubilized with DDM and digitonin (Dig). Source data are available online for this figure.

    Techniques Used: Generated, Labeling, Western Blot, Immunodetection

    Proteomic analyses of UQCR10 and CYC1‐containing protein associations in Δ4‐CYB cells. See also Fig EV3 SDS–PAGE, Western blot, and immunodetection, with the indicated specific antibodies, of Δ4‐CYB and WT cells expressing HA‐tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty). BNGE, Western blot, and immunodetection, with an anti‐HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM. BNGE, Western blot, and immunodetection, with the monoclonal (M) anti‐UQCRQ antibody (Abcam ab110255), of non‐transduced Δ4‐CYB and WT cells. The mitoplast samples were solubilized with DDM (See also Fig EV1). Scatter plot generated from the analysis of the logarithmic heavy (H)‐to‐light (L) ratios in the x ‐axis and the reverse in the y ‐axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti‐HA immunopurification of Δ4‐CYB and WT cells expressing UQCR10 HA . Complexome profiles, generated as in Fig 3 , for the proteins found specifically enriched in Δ4‐CYB UQCR10 HA , according to the SILAC immunopurification experiments shown in (D). The represented values are the mean ± SEM of the two reciprocal labeling experiments. SDS–PAGE, Western blot, and immunodetection, with the indicated specific antibodies, of Δ4‐CYB and WT cells expressing an HA‐tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty). BNGE, Western blot, and immunodetection, with an anti‐HA tag antibody, of samples from the same cell lines as in (F) solubilized either with digitonin. Scatter plot generated from the analysis of the logarithmic heavy (H)‐to‐light (L) ratios in the x ‐axis and the reverse in the y ‐axis, in the two reciprocal labeling SILAC experiments (A and B) of anti‐HA immunopurification of Δ4‐CYB and WT cells expressing CYC1 HA . Source data are available online for this figure.
    Figure Legend Snippet: Proteomic analyses of UQCR10 and CYC1‐containing protein associations in Δ4‐CYB cells. See also Fig EV3 SDS–PAGE, Western blot, and immunodetection, with the indicated specific antibodies, of Δ4‐CYB and WT cells expressing HA‐tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty). BNGE, Western blot, and immunodetection, with an anti‐HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM. BNGE, Western blot, and immunodetection, with the monoclonal (M) anti‐UQCRQ antibody (Abcam ab110255), of non‐transduced Δ4‐CYB and WT cells. The mitoplast samples were solubilized with DDM (See also Fig EV1). Scatter plot generated from the analysis of the logarithmic heavy (H)‐to‐light (L) ratios in the x ‐axis and the reverse in the y ‐axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti‐HA immunopurification of Δ4‐CYB and WT cells expressing UQCR10 HA . Complexome profiles, generated as in Fig 3 , for the proteins found specifically enriched in Δ4‐CYB UQCR10 HA , according to the SILAC immunopurification experiments shown in (D). The represented values are the mean ± SEM of the two reciprocal labeling experiments. SDS–PAGE, Western blot, and immunodetection, with the indicated specific antibodies, of Δ4‐CYB and WT cells expressing an HA‐tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty). BNGE, Western blot, and immunodetection, with an anti‐HA tag antibody, of samples from the same cell lines as in (F) solubilized either with digitonin. Scatter plot generated from the analysis of the logarithmic heavy (H)‐to‐light (L) ratios in the x ‐axis and the reverse in the y ‐axis, in the two reciprocal labeling SILAC experiments (A and B) of anti‐HA immunopurification of Δ4‐CYB and WT cells expressing CYC1 HA . Source data are available online for this figure.

    Techniques Used: SDS Page, Western Blot, Immunodetection, Expressing, Transduction, Plasmid Preparation, Generated, Labeling, Immu-Puri

    Reduced steady‐state levels of structural MRC subunits in Δ4‐CYB cells Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy‐to‐light (H/L) and light‐to‐heavy (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and Δ4‐CYB cells (see also Fig EV1 ). The logarithmic ratios were calculated using MaxQuant (Cox Mann, 2008 ), and the statistical significance of the differences for the enrichment or depletion of the proteins was determined with Perseus (Cox Mann, 2011 ; Tyanova et al , 2016 ). Labeling of the thirteen mtDNA‐encoded MRC structural subunits. Cells were incubated with [ 35 S]‐L‐Met for 1 h in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation. Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD ( n = 3). Two‐way ANOVA with Sidak's multiple comparisons test **** P
    Figure Legend Snippet: Reduced steady‐state levels of structural MRC subunits in Δ4‐CYB cells Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy‐to‐light (H/L) and light‐to‐heavy (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and Δ4‐CYB cells (see also Fig EV1 ). The logarithmic ratios were calculated using MaxQuant (Cox Mann, 2008 ), and the statistical significance of the differences for the enrichment or depletion of the proteins was determined with Perseus (Cox Mann, 2011 ; Tyanova et al , 2016 ). Labeling of the thirteen mtDNA‐encoded MRC structural subunits. Cells were incubated with [ 35 S]‐L‐Met for 1 h in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation. Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD ( n = 3). Two‐way ANOVA with Sidak's multiple comparisons test **** P

    Techniques Used: Generated, Mass Spectrometry, Labeling, Isolation, Incubation, Immunodetection, Western Blot, SDS Page

    Alternative oxidase (AOX) expression and function in WT and Δ4‐CYB cells SDS–PAGE, Western blot, and immunodetection of AOX HA expression in whole‐cell lysates from WT and Δ4‐CYB cells transduced with the AOX HA /pWPXLd‐ires‐Hygro R lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd‐ires‐Hygro R vector (EV). Growth curves of the AOX HA expressing cell lines and their corresponding EV controls. Cell growth was monitored every 6 h after substituting the medium in two replicate 24‐well plates, one plate with medium without uridine (Uridine − ), and the second plate with medium supplemented with 50 μg/ml uridine (Uridine + ). The graphs show the average confluence ± SD at each time point ( n = 6 wells per cell line). Immunodetection of cI structural subunits and NDUFAF2 in the same samples as in panel (A). 1D BNGE, Western blot, and immunodetection analyses of digitonin‐solubilized mitochondria from WT and Δ4‐CYB cybrids expressing AOX HA and their corresponding EV controls. Complex I in‐gel activity assays (IGA) after BNGE as in panel (D). The gels were incubated in the IGA reaction mixture for 5 h. The asterisk indicates the presence of a high‐molecular‐weight cI‐containing band of unknown nature (see main text). Spectrophotometric kinetic measurements of cI activity in WT and Δ4‐CYB AOX HA and EV samples normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD ( n = 5 biological replicates). Two‐way ANOVA with Tukey's multiple comparisons test ** P = 0.0077 (WT AOX HA versus Δ4‐CYB AOX HA ); ** P = 0.0044 (Δ4‐CYB EV versus Δ4‐CYB AOX HA ); *** P = 0.0003; **** P
    Figure Legend Snippet: Alternative oxidase (AOX) expression and function in WT and Δ4‐CYB cells SDS–PAGE, Western blot, and immunodetection of AOX HA expression in whole‐cell lysates from WT and Δ4‐CYB cells transduced with the AOX HA /pWPXLd‐ires‐Hygro R lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd‐ires‐Hygro R vector (EV). Growth curves of the AOX HA expressing cell lines and their corresponding EV controls. Cell growth was monitored every 6 h after substituting the medium in two replicate 24‐well plates, one plate with medium without uridine (Uridine − ), and the second plate with medium supplemented with 50 μg/ml uridine (Uridine + ). The graphs show the average confluence ± SD at each time point ( n = 6 wells per cell line). Immunodetection of cI structural subunits and NDUFAF2 in the same samples as in panel (A). 1D BNGE, Western blot, and immunodetection analyses of digitonin‐solubilized mitochondria from WT and Δ4‐CYB cybrids expressing AOX HA and their corresponding EV controls. Complex I in‐gel activity assays (IGA) after BNGE as in panel (D). The gels were incubated in the IGA reaction mixture for 5 h. The asterisk indicates the presence of a high‐molecular‐weight cI‐containing band of unknown nature (see main text). Spectrophotometric kinetic measurements of cI activity in WT and Δ4‐CYB AOX HA and EV samples normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD ( n = 5 biological replicates). Two‐way ANOVA with Tukey's multiple comparisons test ** P = 0.0077 (WT AOX HA versus Δ4‐CYB AOX HA ); ** P = 0.0044 (Δ4‐CYB EV versus Δ4‐CYB AOX HA ); *** P = 0.0003; **** P

    Techniques Used: Expressing, SDS Page, Western Blot, Immunodetection, Transduction, Plasmid Preparation, Selection, Transfection, Activity Assay, Incubation, Molecular Weight

    Complex I assembly kinetics in Δ4‐CYB cells SDS–PAGE resolving the radioactively labeled mitochondrial translation products after a 2‐h pulse (P). The 35 S‐Met and the cycloheximide were removed from the medium, and cells were collected at the indicated chase times (2, 5, and 24 h). First‐dimension (1D) BNGE analysis of the same cells as in (A), prepared with digitonin. Denaturing second‐dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species. Complex I‐IGA (cI‐IGA) analysis of digitonin‐solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0 h). After removing the drug and restoring synthesis of the mtDNA‐encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 h. SS = steady state. The asterisk indicates the presence of a high‐molecular‐weight cI‐containing band of unknown nature (see main text). 2D BNGE, Western blot, and immunodetection analysis of WT and Δ4‐CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear‐encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 s. (low exposures) or 160 s. (high exposures) in order to visualize the qualitative signals in the Δ4‐CYB samples. Source data are available online for this figure.
    Figure Legend Snippet: Complex I assembly kinetics in Δ4‐CYB cells SDS–PAGE resolving the radioactively labeled mitochondrial translation products after a 2‐h pulse (P). The 35 S‐Met and the cycloheximide were removed from the medium, and cells were collected at the indicated chase times (2, 5, and 24 h). First‐dimension (1D) BNGE analysis of the same cells as in (A), prepared with digitonin. Denaturing second‐dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species. Complex I‐IGA (cI‐IGA) analysis of digitonin‐solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0 h). After removing the drug and restoring synthesis of the mtDNA‐encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 h. SS = steady state. The asterisk indicates the presence of a high‐molecular‐weight cI‐containing band of unknown nature (see main text). 2D BNGE, Western blot, and immunodetection analysis of WT and Δ4‐CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear‐encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 s. (low exposures) or 160 s. (high exposures) in order to visualize the qualitative signals in the Δ4‐CYB samples. Source data are available online for this figure.

    Techniques Used: SDS Page, Labeling, Incubation, Molecular Weight, Western Blot, Immunodetection

    40) Product Images from "A Hyaluronan-binding Peptide (P15-1) Reduces Inflammatory and Catabolic Events in IL-1β-treated Human Articular Chondrocytes"

    Article Title: A Hyaluronan-binding Peptide (P15-1) Reduces Inflammatory and Catabolic Events in IL-1β-treated Human Articular Chondrocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-57586-7

    ( A) Immunoblot analysis of cell extracts from human articular chondrocytes treated with IL-1β, IL-1β and P15-1, or IL-1β, P15-1 and HMWHA for 15 min or 48 h with antibodies specific for phosphorylated (activated) form of p38 (p-p38) or total p38 (p38). ( B) The optical densities of the p-p38 and p38 bands were quantitated by densitometry. Results are expressed as optical density of p-p38 band divided by the optical density of the p38 band and relative units to the value of vehicle-treated cells at 15 min or 48 h, which was set as 1. The blot in ( A) is representative of 3 separate experiments with similar results. The optical densities were analyzed on three immunoblots and values are mean ± SD.
    Figure Legend Snippet: ( A) Immunoblot analysis of cell extracts from human articular chondrocytes treated with IL-1β, IL-1β and P15-1, or IL-1β, P15-1 and HMWHA for 15 min or 48 h with antibodies specific for phosphorylated (activated) form of p38 (p-p38) or total p38 (p38). ( B) The optical densities of the p-p38 and p38 bands were quantitated by densitometry. Results are expressed as optical density of p-p38 band divided by the optical density of the p38 band and relative units to the value of vehicle-treated cells at 15 min or 48 h, which was set as 1. The blot in ( A) is representative of 3 separate experiments with similar results. The optical densities were analyzed on three immunoblots and values are mean ± SD.

    Techniques Used: Western Blot

    Related Articles

    MTT Assay:

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    Immunohistochemistry:

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    Affinity Precipitation:

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    Expressing:

    Article Title: Phosphatidylethanolamine made in the inner mitochondrial membrane is essential for yeast cytochrome bc1 complex function
    Article Snippet: .. Expression of Cox8-His was verified in yeast and mitochondrial lysates by immunoblot using a 6 × -His monoclonal antibody (His.H8, Thermofisher). ..

    Article Title: Phenethyl isothiocyanate Triggers Apoptosis, Combats Oxidative Stress and Inhibits Growth of Ehrlich Ascites Carcinoma Mouse Model
    Article Snippet: .. Quantitative, Real-Time Polymerase Chain Reaction (RT-PCR) for Bax, caspase-9 and Bcl-2 gene expression Total RNA was isolated from EAC cells using TRIzol® Reagent (Ambion, Life Technologies, USA) according to the manufacturer’s instructions. .. The quantity and quality of the isolated RNA was assessed spectrophotometrically at 260 nm and 260/280 nm ratio, respectively, using NanoPhotometer® (Implen, GmbH, Germany).

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    Microarray:

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    Isolation:

    Article Title: Phenethyl isothiocyanate Triggers Apoptosis, Combats Oxidative Stress and Inhibits Growth of Ehrlich Ascites Carcinoma Mouse Model
    Article Snippet: .. Quantitative, Real-Time Polymerase Chain Reaction (RT-PCR) for Bax, caspase-9 and Bcl-2 gene expression Total RNA was isolated from EAC cells using TRIzol® Reagent (Ambion, Life Technologies, USA) according to the manufacturer’s instructions. .. The quantity and quality of the isolated RNA was assessed spectrophotometrically at 260 nm and 260/280 nm ratio, respectively, using NanoPhotometer® (Implen, GmbH, Germany).

    CTL Assay:

    Article Title: Glycan Elongation Beyond the Mucin Associated Tn Antigen Protects Tumor Cells from Immune-Mediated Killing
    Article Snippet: .. ADCC and CTL-mediated cytotoxicity Capan-1 and T47D cells were used as target cells in the ADCC assay after trypsinizing with TripLE Express (Gibco. .. 12605-028) at 90% confluence, and were transferred into 6 well plates with 0.8*106 cells/well with the addition of 2.5 ug/ml of Erbitux® or irrelevant human IgG and 4*106 purified monocytes when indicated (in shaving experiments).

    Real-time Polymerase Chain Reaction:

    Article Title: Phenethyl isothiocyanate Triggers Apoptosis, Combats Oxidative Stress and Inhibits Growth of Ehrlich Ascites Carcinoma Mouse Model
    Article Snippet: .. Quantitative, Real-Time Polymerase Chain Reaction (RT-PCR) for Bax, caspase-9 and Bcl-2 gene expression Total RNA was isolated from EAC cells using TRIzol® Reagent (Ambion, Life Technologies, USA) according to the manufacturer’s instructions. .. The quantity and quality of the isolated RNA was assessed spectrophotometrically at 260 nm and 260/280 nm ratio, respectively, using NanoPhotometer® (Implen, GmbH, Germany).

    Activation Assay:

    Article Title: Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells
    Article Snippet: .. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, TRIzol® reagent, and caspase-9 and caspase-3 activation kits were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA). .. Monoclonal antibodies against Bcl-2, Bcl2-associated X protein (Bax), cyclin D1 and cyclin-dependent kinase 4 (CDK4) and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

    Migration:

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    ADCC Assay:

    Article Title: Glycan Elongation Beyond the Mucin Associated Tn Antigen Protects Tumor Cells from Immune-Mediated Killing
    Article Snippet: .. ADCC and CTL-mediated cytotoxicity Capan-1 and T47D cells were used as target cells in the ADCC assay after trypsinizing with TripLE Express (Gibco. .. 12605-028) at 90% confluence, and were transferred into 6 well plates with 0.8*106 cells/well with the addition of 2.5 ug/ml of Erbitux® or irrelevant human IgG and 4*106 purified monocytes when indicated (in shaving experiments).

    Modification:

    Article Title: Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells
    Article Snippet: .. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, TRIzol® reagent, and caspase-9 and caspase-3 activation kits were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA). .. Monoclonal antibodies against Bcl-2, Bcl2-associated X protein (Bax), cyclin D1 and cyclin-dependent kinase 4 (CDK4) and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

    Staining:

    Article Title: CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2
    Article Snippet: .. Plasmids and antibodies, protein interaction assays and immunoblot, gene expression analysis by Affymetrix microarray, MTT, migration, invasion, colony formation assays, xenograft experiments, immunohistochemical staining, DNA affinity precipitation (DAPA) assays and statistical analysis are explained in detail in Supplementary Data . .. CCAR1 functions as an AR co-activator Given our findings that CCAR1 interacts with members of the NR family , such as ERα and glucocorticoid receptor (GR), we first examined the association between endogenous CCAR1 and AR in AR-positive LNCaP prostate cancer cells by coimmunoprecipitation (CoIP) assays.

    Reverse Transcription Polymerase Chain Reaction:

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  • 99
    Thermo Fisher nupage bis tris sds page gel
    In vitro translation of dihydrofolate reductase ( DHFR ) in the presence of synthetic peptides representing the toxin and antitoxin proteins of the D11S_1718‐1719 and D11S_1194‐1195 toxin‐antitoxin ( TA ) systems. In vitro translation reactions were incubated with the toxin or antitoxin proteins of the TA systems, either alone or in combination and reaction products were electrophoresed in a <t>Tris‐Glycine</t> <t>SDS</t> ‐ <t>PAGE</t> gel. Lanes 1 and 8, positive control reaction without toxin or antitoxin protein (arrows indicates the DHFR protein); Lane 2, D11S_1194 toxin only; Lane 3, D11S_1195 antitoxin only; Lane 4, equal mixture of the D11S_1194‐1195 toxin and antitoxin; Lane 5, D11S_1718 toxin only; Lane 6, D11S_1719 antitoxin only; Lane 7, equal mixture of the D11S_1718‐1719 toxin and antitoxin; Lane 9, empty; Lane 10, size markers
    Nupage Bis Tris Sds Page Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pre cast sds page gel
    Activation of WalK autophosphorylation by YycH and YycI in phospholipid liposomes. WalK was reconstituted separately and with increasing amounts of YycH and/or YycI into phospholipid liposomes with molar ratios of 2:1, 2:2, and 2:4 for WalK(monomer)/YycH and WalK/YycI and 2:1:1, 2:2:2, and 2:4:4 for WalK/YycH/YycI. Autophosphorylation was started using [γ 32 P]-ATP and liposomes were loaded on a 10% <t>SDS-PAGE</t> gel. WalK with bound radiolabeled phosphate was visualized using a storage phosphor screen (top) and the gels were stained with Coomassie afterwards (bottom). Representative autoradiograms and Coomassie stained gels of three different experiments are shown. YycH and YycI increased the autophosphorylation of WalK, especially when both regulatory proteins were present in the liposomes.
    Pre Cast Sds Page Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher vrc01 n
    Median-effect model analysis of efficacy in vaginal HIV-1 prevention using a triple drug combination ( N  = 10 per dosing group, 5 groups per study).  F a , fraction affected;  F u , fraction unaffected;  D , dose (nM). For panels ( A – C ) Blue, TDF; red, FTC, green; VRC01- N ; orange, TDF-FTC-VRC01- N . ( A ) log–log dose–response relationships summarized in Table   1 . ( B ) Combination index ( CI ) plot for TDF-FTC-VRC01- N.  Open circles correspond to datapoints from separate experiment.  CI >  1 antagonism;  CI  = 1 (broken line), additive effect;  CI
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    Image Search Results


    In vitro translation of dihydrofolate reductase ( DHFR ) in the presence of synthetic peptides representing the toxin and antitoxin proteins of the D11S_1718‐1719 and D11S_1194‐1195 toxin‐antitoxin ( TA ) systems. In vitro translation reactions were incubated with the toxin or antitoxin proteins of the TA systems, either alone or in combination and reaction products were electrophoresed in a Tris‐Glycine SDS ‐ PAGE gel. Lanes 1 and 8, positive control reaction without toxin or antitoxin protein (arrows indicates the DHFR protein); Lane 2, D11S_1194 toxin only; Lane 3, D11S_1195 antitoxin only; Lane 4, equal mixture of the D11S_1194‐1195 toxin and antitoxin; Lane 5, D11S_1718 toxin only; Lane 6, D11S_1719 antitoxin only; Lane 7, equal mixture of the D11S_1718‐1719 toxin and antitoxin; Lane 9, empty; Lane 10, size markers

    Journal: Molecular Oral Microbiology

    Article Title: Identification and functional characterization of type II toxin/antitoxin systems in Aggregatibacter actinomycetemcomitans. Identification and functional characterization of type II toxin/antitoxin systems in Aggregatibacter actinomycetemcomitans

    doi: 10.1111/omi.12215

    Figure Lengend Snippet: In vitro translation of dihydrofolate reductase ( DHFR ) in the presence of synthetic peptides representing the toxin and antitoxin proteins of the D11S_1718‐1719 and D11S_1194‐1195 toxin‐antitoxin ( TA ) systems. In vitro translation reactions were incubated with the toxin or antitoxin proteins of the TA systems, either alone or in combination and reaction products were electrophoresed in a Tris‐Glycine SDS ‐ PAGE gel. Lanes 1 and 8, positive control reaction without toxin or antitoxin protein (arrows indicates the DHFR protein); Lane 2, D11S_1194 toxin only; Lane 3, D11S_1195 antitoxin only; Lane 4, equal mixture of the D11S_1194‐1195 toxin and antitoxin; Lane 5, D11S_1718 toxin only; Lane 6, D11S_1719 antitoxin only; Lane 7, equal mixture of the D11S_1718‐1719 toxin and antitoxin; Lane 9, empty; Lane 10, size markers

    Article Snippet: Synthetic peptides were dissolved in 0.05 m phosphate buffer, pH 7.8, containing 300 mm NaCl and 0.01% trifluoroacetic acid and peptide purity and size was confirmed using a NuPAGE Bis‐Tris SDS‐PAGE gel (Thermo Fisher Scientific, Waltham, MA).

    Techniques: In Vitro, Incubation, SDS Page, Positive Control

    Activation of WalK autophosphorylation by YycH and YycI in phospholipid liposomes. WalK was reconstituted separately and with increasing amounts of YycH and/or YycI into phospholipid liposomes with molar ratios of 2:1, 2:2, and 2:4 for WalK(monomer)/YycH and WalK/YycI and 2:1:1, 2:2:2, and 2:4:4 for WalK/YycH/YycI. Autophosphorylation was started using [γ 32 P]-ATP and liposomes were loaded on a 10% SDS-PAGE gel. WalK with bound radiolabeled phosphate was visualized using a storage phosphor screen (top) and the gels were stained with Coomassie afterwards (bottom). Representative autoradiograms and Coomassie stained gels of three different experiments are shown. YycH and YycI increased the autophosphorylation of WalK, especially when both regulatory proteins were present in the liposomes.

    Journal: Microorganisms

    Article Title: YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation

    doi: 10.3390/microorganisms8060870

    Figure Lengend Snippet: Activation of WalK autophosphorylation by YycH and YycI in phospholipid liposomes. WalK was reconstituted separately and with increasing amounts of YycH and/or YycI into phospholipid liposomes with molar ratios of 2:1, 2:2, and 2:4 for WalK(monomer)/YycH and WalK/YycI and 2:1:1, 2:2:2, and 2:4:4 for WalK/YycH/YycI. Autophosphorylation was started using [γ 32 P]-ATP and liposomes were loaded on a 10% SDS-PAGE gel. WalK with bound radiolabeled phosphate was visualized using a storage phosphor screen (top) and the gels were stained with Coomassie afterwards (bottom). Representative autoradiograms and Coomassie stained gels of three different experiments are shown. YycH and YycI increased the autophosphorylation of WalK, especially when both regulatory proteins were present in the liposomes.

    Article Snippet: The reaction was stopped with 2x Laemmli SDS sample buffer and loaded onto a pre-cast SDS-PAGE gel (NuPAGE Bis Tris Gels, Thermo Fisher Scientific).

    Techniques: Activation Assay, SDS Page, Staining

    Median-effect model analysis of efficacy in vaginal HIV-1 prevention using a triple drug combination ( N  = 10 per dosing group, 5 groups per study).  F a , fraction affected;  F u , fraction unaffected;  D , dose (nM). For panels ( A – C ) Blue, TDF; red, FTC, green; VRC01- N ; orange, TDF-FTC-VRC01- N . ( A ) log–log dose–response relationships summarized in Table   1 . ( B ) Combination index ( CI ) plot for TDF-FTC-VRC01- N.  Open circles correspond to datapoints from separate experiment.  CI >  1 antagonism;  CI  = 1 (broken line), additive effect;  CI

    Journal: Scientific Reports

    Article Title: Highly synergistic drug combination prevents vaginal HIV infection in humanized mice

    doi: 10.1038/s41598-020-69937-5

    Figure Lengend Snippet: Median-effect model analysis of efficacy in vaginal HIV-1 prevention using a triple drug combination ( N  = 10 per dosing group, 5 groups per study). F a , fraction affected; F u , fraction unaffected; D , dose (nM). For panels ( A – C ) Blue, TDF; red, FTC, green; VRC01- N ; orange, TDF-FTC-VRC01- N . ( A ) log–log dose–response relationships summarized in Table 1 . ( B ) Combination index ( CI ) plot for TDF-FTC-VRC01- N. Open circles correspond to datapoints from separate experiment. CI >  1 antagonism; CI  = 1 (broken line), additive effect; CI

    Article Snippet: A predetermined amount of VRC01-N was electrophoresed on a Novex NuPAGE 4–12% Bis–Tris protein gel (NP0321BOX, ThermoFisher Scientific, Waltham, MA) with MES SDS Running buffer (NP0002, ThermoFisher Scientific) using an XCell SureLock Electrophoresis System (EI0002, ThermoFisher Scientific) and a Novex Sharp Pre-Stained Protein Standard (LC5800, ThermoFisher Scientific).

    Techniques:

    Dose–response curves for vaginal HIV-1 challenge studies in humanized BLT mice. Plots of efficacy  versus  dose of ( A ) VRC01- N  ( N  = 8 per dosing group, 5 groups) and ( B ) TDF-FTC-VRC01- N  combination ( N  = 10 per dosing group, 5 groups per study; two studies) applied prior to HIV-1 challenge. Open circle corresponds to datapoint from separate experiment. Dashed lines are fits to a sigmoidal dose–response (variable slope) model used to calculate EC 50  of the drug or drug combination providing protection against vaginal HIV-1 challenges.

    Journal: Scientific Reports

    Article Title: Highly synergistic drug combination prevents vaginal HIV infection in humanized mice

    doi: 10.1038/s41598-020-69937-5

    Figure Lengend Snippet: Dose–response curves for vaginal HIV-1 challenge studies in humanized BLT mice. Plots of efficacy versus dose of ( A ) VRC01- N ( N  = 8 per dosing group, 5 groups) and ( B ) TDF-FTC-VRC01- N combination ( N  = 10 per dosing group, 5 groups per study; two studies) applied prior to HIV-1 challenge. Open circle corresponds to datapoint from separate experiment. Dashed lines are fits to a sigmoidal dose–response (variable slope) model used to calculate EC 50 of the drug or drug combination providing protection against vaginal HIV-1 challenges.

    Article Snippet: A predetermined amount of VRC01-N was electrophoresed on a Novex NuPAGE 4–12% Bis–Tris protein gel (NP0321BOX, ThermoFisher Scientific, Waltham, MA) with MES SDS Running buffer (NP0002, ThermoFisher Scientific) using an XCell SureLock Electrophoresis System (EI0002, ThermoFisher Scientific) and a Novex Sharp Pre-Stained Protein Standard (LC5800, ThermoFisher Scientific).

    Techniques: Mouse Assay