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    Millipore optiprep density gradient medium
    The micro RNA ‐generating complex localizes at the neuronal endoplasmic reticulum Components of the RLC co‐precipitate with ER markers in density gradient fractionation. The postnuclear supernatant of cortical neurons was loaded underneath a continuous <t>Optiprep</t> density gradient. ER markers, calnexin, ribophorin I, and Climp63, co‐sedimented with ribosomes (RPS6, ribosomal protein subunit 6 ), mitochondria (Tim23), and RLC proteins at fractions 3–5. The bottom graph shows the density of each fraction. TRBP, PACT, and Ago2 partially co‐localize with the ER marker mCherry‐CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry‐CLIMP63 were immunostained with TRBP, PACT, or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10 μm). GFP‐Dicer partially co‐localizes with mCherry‐CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150 nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2 μm or 10 μm). The majority of Dicer, TRBP, and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (** P
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    The micro RNA ‐generating complex localizes at the neuronal endoplasmic reticulum Components of the RLC co‐precipitate with ER markers in density gradient fractionation. The postnuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers, calnexin, ribophorin I, and Climp63, co‐sedimented with ribosomes (RPS6, ribosomal protein subunit 6 ), mitochondria (Tim23), and RLC proteins at fractions 3–5. The bottom graph shows the density of each fraction. TRBP, PACT, and Ago2 partially co‐localize with the ER marker mCherry‐CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry‐CLIMP63 were immunostained with TRBP, PACT, or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10 μm). GFP‐Dicer partially co‐localizes with mCherry‐CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150 nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2 μm or 10 μm). The majority of Dicer, TRBP, and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (** P

    Journal: EMBO Reports

    Article Title: The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

    doi: 10.15252/embr.201744853

    Figure Lengend Snippet: The micro RNA ‐generating complex localizes at the neuronal endoplasmic reticulum Components of the RLC co‐precipitate with ER markers in density gradient fractionation. The postnuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers, calnexin, ribophorin I, and Climp63, co‐sedimented with ribosomes (RPS6, ribosomal protein subunit 6 ), mitochondria (Tim23), and RLC proteins at fractions 3–5. The bottom graph shows the density of each fraction. TRBP, PACT, and Ago2 partially co‐localize with the ER marker mCherry‐CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry‐CLIMP63 were immunostained with TRBP, PACT, or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10 μm). GFP‐Dicer partially co‐localizes with mCherry‐CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150 nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2 μm or 10 μm). The majority of Dicer, TRBP, and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (** P

    Article Snippet: Nuclei were pelleted by centrifugation 700 × g , 10 min, and the postnuclear supernatant (PNS) was mixed with 50% Optiprep density gradient medium (D1556, Sigma) to make a 35% solution and loaded underneath a 2.5–30% continuous Optiprep gradient.

    Techniques: Fractionation, Marker, Expressing, Fluorescence, Microscopy

    Design and implementation of the approach. ( a ) Schematic of the STAMP (Specifically TArgeted Membrane nanoParticle) approach. In the first step, a small biotin label (yellow) is attached via a spacer arm (orange) to a mitochondrial model preprotein (red). Subsequently, the labelled preprotein is targeted to the mitochondrial import machinery and arrested by means of a tightly folded DHFR domain as a translocation intermediate spanning both TOM (blue) and TIM23 (cyan) complexes. In the third step, the DHFR-linked biotin is bound by streptavidin (purple)-conjugated QDs (black sphere with green spacer arms). The total distance from the QD to the outer mitochondrial membrane is ~10 nm. ( b ) The design of the mitochondrial model preprotein is based on the fusion protein b 2 Δ-DHFR 18 . To enable site-specific biotinylation, endogenous cysteines (C14, C86 and C157) were substituted with serine and a unique cysteine replaced the C-terminal residue D337. ( c ) Blue native electrophoresis and western blot analysis of the preprotein-tethered TOM–TIM23 supercomplex detected with an antibody against Tim23. Note that the TIM23 core complexes (TIM23 CORE ) are quantitatively shifted into TOM–TIM23 supercomplexes on addition of b 2 Δ-DHFR biotin , indicating that the import sites are occupied by preproteins under these conditions. The experiment was repeated three times. ( d ) Free QDs were separated from labelled mitochondria on an OptiPrep gradient. Under white light (tubes 1 and 3) mitochondrial membranes are visible (M, yellow boxes), and under UV excitation, QD 525 is detected (tubes 2 and 4; green boxes). QD 525 co-localization with mitochondria is seen in tube 4.

    Journal: Nature Communications

    Article Title: Visualizing active membrane protein complexes by electron cryotomography

    doi: 10.1038/ncomms5129

    Figure Lengend Snippet: Design and implementation of the approach. ( a ) Schematic of the STAMP (Specifically TArgeted Membrane nanoParticle) approach. In the first step, a small biotin label (yellow) is attached via a spacer arm (orange) to a mitochondrial model preprotein (red). Subsequently, the labelled preprotein is targeted to the mitochondrial import machinery and arrested by means of a tightly folded DHFR domain as a translocation intermediate spanning both TOM (blue) and TIM23 (cyan) complexes. In the third step, the DHFR-linked biotin is bound by streptavidin (purple)-conjugated QDs (black sphere with green spacer arms). The total distance from the QD to the outer mitochondrial membrane is ~10 nm. ( b ) The design of the mitochondrial model preprotein is based on the fusion protein b 2 Δ-DHFR 18 . To enable site-specific biotinylation, endogenous cysteines (C14, C86 and C157) were substituted with serine and a unique cysteine replaced the C-terminal residue D337. ( c ) Blue native electrophoresis and western blot analysis of the preprotein-tethered TOM–TIM23 supercomplex detected with an antibody against Tim23. Note that the TIM23 core complexes (TIM23 CORE ) are quantitatively shifted into TOM–TIM23 supercomplexes on addition of b 2 Δ-DHFR biotin , indicating that the import sites are occupied by preproteins under these conditions. The experiment was repeated three times. ( d ) Free QDs were separated from labelled mitochondria on an OptiPrep gradient. Under white light (tubes 1 and 3) mitochondrial membranes are visible (M, yellow boxes), and under UV excitation, QD 525 is detected (tubes 2 and 4; green boxes). QD 525 co-localization with mitochondria is seen in tube 4.

    Article Snippet: Separation of excess QDs was performed with an OptiPrep density gradient (Sigma-Aldrich, St. Louis, USA), using gradients with 10 steps of 200 μl each, ranging from 0 to 27% iodixanol in ultra-clear centrifuge tubes (Beckman Coulter, Pasadena, USA).

    Techniques: Translocation Assay, Electrophoresis, Western Blot

    Specific labelling determines the total number of preprotein import sites and the degree of clustering. ( a ) Free QDs were separated from labelled mitochondria on an OptiPrep gradient. Under white light (tubes 1 and 3) mitochondria are visible (M, yellow boxes), and under UV excitation, QD 605 is detected (tubes 2 and 4; green boxes). QD 605 co-localization with mitochondria is seen in tube 4. The experiment was repeated four times. ( b ) Confocal fluorescence images of non-importing and importing mitochondria. The mitochondria are labelled with MitoTracker Green and QD 605 fluorescence is shown in red; scale bar, 1 μm. The black space between mitochondria has been removed (original in Supplementary Fig. 5a ). The statistics described in the text were calculated based on n =350 for the control and n =140 for the actively importing mitochondria. ( c ) A slice near the top of the tomogram reveals the location of a cluster of QDs (green arrowheads) on the mitochondrial surface. A slice through the centre shows the position of the cluster with respect to the CJs (yellow arrowheads); scale bar, 100 nm, cluster measures ~80 × 60 nm. ( d ) Segmentation of the volume depicts the three-dimensional distribution of QDs (green spheres) around the mitochondrion relative to the outer membrane (blue) and the cristae (yellow). ( e ) A model of an ellipsoid was used to calculate the surface area of mitochondria based on a , b and c radii as depicted. ( f ) A line graph shows the correlation between mitochondrial size and the number of preprotein import sites, n =12 mitochondria as seen in Table 2 . ( g ) Averaged histogram showing the closest distance between two QDs, calculated from 12 mitochondrial samples ( Supplementary Fig. 6 ) accumulating 1,159 QD 605 data points in total. Error bars indicate the s.d. of the frequency distribution for each minimal distance.

    Journal: Nature Communications

    Article Title: Visualizing active membrane protein complexes by electron cryotomography

    doi: 10.1038/ncomms5129

    Figure Lengend Snippet: Specific labelling determines the total number of preprotein import sites and the degree of clustering. ( a ) Free QDs were separated from labelled mitochondria on an OptiPrep gradient. Under white light (tubes 1 and 3) mitochondria are visible (M, yellow boxes), and under UV excitation, QD 605 is detected (tubes 2 and 4; green boxes). QD 605 co-localization with mitochondria is seen in tube 4. The experiment was repeated four times. ( b ) Confocal fluorescence images of non-importing and importing mitochondria. The mitochondria are labelled with MitoTracker Green and QD 605 fluorescence is shown in red; scale bar, 1 μm. The black space between mitochondria has been removed (original in Supplementary Fig. 5a ). The statistics described in the text were calculated based on n =350 for the control and n =140 for the actively importing mitochondria. ( c ) A slice near the top of the tomogram reveals the location of a cluster of QDs (green arrowheads) on the mitochondrial surface. A slice through the centre shows the position of the cluster with respect to the CJs (yellow arrowheads); scale bar, 100 nm, cluster measures ~80 × 60 nm. ( d ) Segmentation of the volume depicts the three-dimensional distribution of QDs (green spheres) around the mitochondrion relative to the outer membrane (blue) and the cristae (yellow). ( e ) A model of an ellipsoid was used to calculate the surface area of mitochondria based on a , b and c radii as depicted. ( f ) A line graph shows the correlation between mitochondrial size and the number of preprotein import sites, n =12 mitochondria as seen in Table 2 . ( g ) Averaged histogram showing the closest distance between two QDs, calculated from 12 mitochondrial samples ( Supplementary Fig. 6 ) accumulating 1,159 QD 605 data points in total. Error bars indicate the s.d. of the frequency distribution for each minimal distance.

    Article Snippet: Separation of excess QDs was performed with an OptiPrep density gradient (Sigma-Aldrich, St. Louis, USA), using gradients with 10 steps of 200 μl each, ranging from 0 to 27% iodixanol in ultra-clear centrifuge tubes (Beckman Coulter, Pasadena, USA).

    Techniques: Fluorescence

    EHEC O157 OMVs carry a cocktail of virulence factors. (A) Electron microscopy of ultrathin cryosections of LB agar cultures of strains 5791/99 and 493/89 stained with anti- E . coli O157 LPS antibody and Protein A Gold or Protein A Gold alone (control). Examples of OMVs (v) and bacteria (b) are indicated. Frames depict OMVs located in other microscopic fields than the producing bacteria. Scale bars are 150 nm. (B) Distribution of virulence factors in OMVs and OMV-free supernatants determined by immunoblot with antibodies against OmpA (an OMV marker) and the indicated virulence proteins. (C) Distribution of virulence factors in OptiPrep density gradient fractions (1 to 12, collected from top to bottom) of O157 OMVs determined by immunoblot. The lanes designated OMV contain non-fractionated OMVs (positive control). (D) Localizations of virulence factors within 5791/99 OMVs visualized by electron microscopy of ultrathin cryosections of OptipPrep-purified OMVs stained with antibodies against the indicated virulence factors and Protein A Gold (panels 1 and 2) or Protein A Gold alone (panels 3; control). Bars are 100 nm. Note that by electron microscopy of immunostained ultrathin cryosections only 10%–15% of the total antigen present in the section can be detected [ 31 ] explaining relatively low numbers of signals observed for most virulence factors. (E) Immunoblots of proteinase K (PK)-untreated (PK-) and PK-treated (PK+) O157 OMVs either intact (EDTA-) or lysed with 0.1 M EDTA (EDTA+) with the indicated antibodies. (Anti-CdtV-C antibody is not suitable for electron microscopy but detects CdtV-C by immunoblot).

    Journal: PLoS Pathogens

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

    doi: 10.1371/journal.ppat.1006159

    Figure Lengend Snippet: EHEC O157 OMVs carry a cocktail of virulence factors. (A) Electron microscopy of ultrathin cryosections of LB agar cultures of strains 5791/99 and 493/89 stained with anti- E . coli O157 LPS antibody and Protein A Gold or Protein A Gold alone (control). Examples of OMVs (v) and bacteria (b) are indicated. Frames depict OMVs located in other microscopic fields than the producing bacteria. Scale bars are 150 nm. (B) Distribution of virulence factors in OMVs and OMV-free supernatants determined by immunoblot with antibodies against OmpA (an OMV marker) and the indicated virulence proteins. (C) Distribution of virulence factors in OptiPrep density gradient fractions (1 to 12, collected from top to bottom) of O157 OMVs determined by immunoblot. The lanes designated OMV contain non-fractionated OMVs (positive control). (D) Localizations of virulence factors within 5791/99 OMVs visualized by electron microscopy of ultrathin cryosections of OptipPrep-purified OMVs stained with antibodies against the indicated virulence factors and Protein A Gold (panels 1 and 2) or Protein A Gold alone (panels 3; control). Bars are 100 nm. Note that by electron microscopy of immunostained ultrathin cryosections only 10%–15% of the total antigen present in the section can be detected [ 31 ] explaining relatively low numbers of signals observed for most virulence factors. (E) Immunoblots of proteinase K (PK)-untreated (PK-) and PK-treated (PK+) O157 OMVs either intact (EDTA-) or lysed with 0.1 M EDTA (EDTA+) with the indicated antibodies. (Anti-CdtV-C antibody is not suitable for electron microscopy but detects CdtV-C by immunoblot).

    Article Snippet: OMV fractionation, dissociation assay, prote inase K (PK) assay OMVs were fractionated by OptiPrep (Sigma-Aldrich) density gradient ultracentrifugation [ ] and gradient fractions were analyzed by immunoblot with anti-OmpA, anti-Stx2a, anti-CdtV-A, -B, and -C, anti-EHEC-Hly, and anti-H7 antibodies.

    Techniques: Electron Microscopy, Staining, Marker, Positive Control, Purification, Western Blot

    Tfn and dIgA transit through the same EE and RE populations. (A) FITC-Tfn and Texas red–dIgA were bound to the basolateral surface of MDCK cells doubly transfected with the TfnR and pIgR at 0°C. The ligands were then internalized at 37°C for 2.5 (top) or 25 min (bottom). Tfn (green) and dIgA (red) were imaged separately and merged; yellow spots indicate likely double-positive structures. 2.5-min images were taken 0.5 μm from the filter surface; 25-min images were taken ∼5 μm from the filter surface, in the apical cytoplasm. Bar, 10 μm. (B) 125 I-Tfn or 125 I-dIgA were bound to the basolateral surface of filter-grown MDCK cells doubly transfected with TfnR and pIgR at 0°C. The ligands were internalized at 37°C for 2.5 min, then the cells were harvested and homogenates were fractionated in parallel on Optiprep density gradients. EE peak is at fraction 29. (C) After 25 min of internalization, both Tfn and dIgA shifted to apparently the same, less dense RE compartment. Closed symbols indicate Tfn, open symbols indicate dIgA.

    Journal: The Journal of Cell Biology

    Article Title: The Receptor Recycling Pathway Contains Two Distinct Populations of Early Endosomes with Different Sorting Functions

    doi:

    Figure Lengend Snippet: Tfn and dIgA transit through the same EE and RE populations. (A) FITC-Tfn and Texas red–dIgA were bound to the basolateral surface of MDCK cells doubly transfected with the TfnR and pIgR at 0°C. The ligands were then internalized at 37°C for 2.5 (top) or 25 min (bottom). Tfn (green) and dIgA (red) were imaged separately and merged; yellow spots indicate likely double-positive structures. 2.5-min images were taken 0.5 μm from the filter surface; 25-min images were taken ∼5 μm from the filter surface, in the apical cytoplasm. Bar, 10 μm. (B) 125 I-Tfn or 125 I-dIgA were bound to the basolateral surface of filter-grown MDCK cells doubly transfected with TfnR and pIgR at 0°C. The ligands were internalized at 37°C for 2.5 min, then the cells were harvested and homogenates were fractionated in parallel on Optiprep density gradients. EE peak is at fraction 29. (C) After 25 min of internalization, both Tfn and dIgA shifted to apparently the same, less dense RE compartment. Closed symbols indicate Tfn, open symbols indicate dIgA.

    Article Snippet: Western Blot Fractions from Optiprep density gradients were mixed with an equal volume of H2 O containing 2% (wt/vol) sodium deoxycholate ( Sigma Chemical Co. ) and vortexed.

    Techniques: Transfection

    Separation of EEs and REs by Optiprep gradient centrifugation. MDCK cells transfected with the human TfnR were grown on Transwell filters. 125 I-Tfn was bound at 0°C and then internalized for either 2.5 or 25 min. The cells were acid washed to remove remaining surface bound 125 I-Tfn, homogenized, and postnuclear supernatants separated on 5–20% linear Optiprep gradients. The low density region of the gradients is on the left (fraction 1). 125 I-Tfn internalized for 2.5 min was contained within membranes which sedimented towards the bottom of the gradient (squares). After 25 min of chase, the Tfn was recovered as a single peak of slightly lower density (circles). The position of the plasma membrane was determined by alkaline phosphodiesterase activity and used to indicate the position of plasma membranes on the gradient. β-Hexosaminidase activity (lysosomes) formed a characteristically well defined peak in fractions 9–15 (not shown). The radioactivity in each fraction was normalized to percent total label loaded in each gradient (191,589 cpm for 2.5-min time point; 40,767 cpm for 25-min time point).

    Journal: The Journal of Cell Biology

    Article Title: The Receptor Recycling Pathway Contains Two Distinct Populations of Early Endosomes with Different Sorting Functions

    doi:

    Figure Lengend Snippet: Separation of EEs and REs by Optiprep gradient centrifugation. MDCK cells transfected with the human TfnR were grown on Transwell filters. 125 I-Tfn was bound at 0°C and then internalized for either 2.5 or 25 min. The cells were acid washed to remove remaining surface bound 125 I-Tfn, homogenized, and postnuclear supernatants separated on 5–20% linear Optiprep gradients. The low density region of the gradients is on the left (fraction 1). 125 I-Tfn internalized for 2.5 min was contained within membranes which sedimented towards the bottom of the gradient (squares). After 25 min of chase, the Tfn was recovered as a single peak of slightly lower density (circles). The position of the plasma membrane was determined by alkaline phosphodiesterase activity and used to indicate the position of plasma membranes on the gradient. β-Hexosaminidase activity (lysosomes) formed a characteristically well defined peak in fractions 9–15 (not shown). The radioactivity in each fraction was normalized to percent total label loaded in each gradient (191,589 cpm for 2.5-min time point; 40,767 cpm for 25-min time point).

    Article Snippet: Western Blot Fractions from Optiprep density gradients were mixed with an equal volume of H2 O containing 2% (wt/vol) sodium deoxycholate ( Sigma Chemical Co. ) and vortexed.

    Techniques: Gradient Centrifugation, Transfection, Activity Assay, Radioactivity

    EEs and REs have distinct protein compositions. MDCK cells cotransfected with human TfnR and pIgR were grown on Transwell filters and crude postnuclear supernatants (including cytosol) were centrifuged using 10–20% Optiprep density gradients. (A) 125 I-Tfn was bound at 0°C and then internalized for 2.5 or 25 min as in Fig. 2 . Postnuclear supernatants were prepared and then centrifuged. Percent total radioactivity in each gradient is indicated for the 2.5-min time point (squares) and the 25-min time point (triangles). 125 I-Tfn peaks around fraction 5 cosediment with the basolateral plasma membrane marker alkaline phosphatase; more 125 I-Tfn was found in this region than in Fig. 2 because cells were not extensively acid washed before homogenization. EEs are distinguished by a peak at fraction 23 and REs by a peak at fraction 20. (B) Western blots of fractionated cells using antibodies to human TfnR, human rab4, and human rab11. Half of the entire volume of each fraction was loaded in each lane. TfnR was found in all fractions but is most abundant in the RE-containing fractions identified by the 25-min 125 I-Tfn peak. rab4 cosedimented most closely with the EE peak; rab11 was relatively depleted from these EE-containing fractions relative to higher density fractions more coincident with REs (TfnR, 25 min 125 I-Tfn). (C) Double label immunoelectron microscopy of endosomes isolated on Optiprep density gradients. Tfn receptor was labeled with 5-nm gold in both images (small arrowheads). Fraction 24 was additionally labeled for rab11 with 10-nm gold (large arrowheads). Fraction 27 was labeled for rab4 with 10-nm gold (large arrowheads).

    Journal: The Journal of Cell Biology

    Article Title: The Receptor Recycling Pathway Contains Two Distinct Populations of Early Endosomes with Different Sorting Functions

    doi:

    Figure Lengend Snippet: EEs and REs have distinct protein compositions. MDCK cells cotransfected with human TfnR and pIgR were grown on Transwell filters and crude postnuclear supernatants (including cytosol) were centrifuged using 10–20% Optiprep density gradients. (A) 125 I-Tfn was bound at 0°C and then internalized for 2.5 or 25 min as in Fig. 2 . Postnuclear supernatants were prepared and then centrifuged. Percent total radioactivity in each gradient is indicated for the 2.5-min time point (squares) and the 25-min time point (triangles). 125 I-Tfn peaks around fraction 5 cosediment with the basolateral plasma membrane marker alkaline phosphatase; more 125 I-Tfn was found in this region than in Fig. 2 because cells were not extensively acid washed before homogenization. EEs are distinguished by a peak at fraction 23 and REs by a peak at fraction 20. (B) Western blots of fractionated cells using antibodies to human TfnR, human rab4, and human rab11. Half of the entire volume of each fraction was loaded in each lane. TfnR was found in all fractions but is most abundant in the RE-containing fractions identified by the 25-min 125 I-Tfn peak. rab4 cosedimented most closely with the EE peak; rab11 was relatively depleted from these EE-containing fractions relative to higher density fractions more coincident with REs (TfnR, 25 min 125 I-Tfn). (C) Double label immunoelectron microscopy of endosomes isolated on Optiprep density gradients. Tfn receptor was labeled with 5-nm gold in both images (small arrowheads). Fraction 24 was additionally labeled for rab11 with 10-nm gold (large arrowheads). Fraction 27 was labeled for rab4 with 10-nm gold (large arrowheads).

    Article Snippet: Western Blot Fractions from Optiprep density gradients were mixed with an equal volume of H2 O containing 2% (wt/vol) sodium deoxycholate ( Sigma Chemical Co. ) and vortexed.

    Techniques: Radioactivity, Marker, Homogenization, Western Blot, Immuno-Electron Microscopy, Isolation, Labeling

    Partial retention of FITC-Tfn in REs by AlF 4 . (A) CHO cells and MDCK cells expressing human TfnR were pulsed with FITC-Tfn for 30 min at 37°C, conditions which labeled both peripheral and perinuclear endosome populations (panels A and B). The cells were then chased in the presence of excess unlabeled Tfn for 30 min (panels C and D). Although a few cells remained brightly labeled, most exhibited a marked loss of FITC-Tfn, with remaining cell-associated FITC-Tfn found predominantly in perinuclear vesicles (arrows). When cells were chased in the presence of unlabeled Tfn and AlF 4 for 30 min, however, FITC-Tfn was largely retained in the perinuclear endosomes (panels E and F). In CHO cells, these structures exhibited the characteristic distribution as a tightly organized array near the microtubule organizing center; in MDCK cells, they were more diffusely distributed, but nevertheless closely apposed to the nucleus. (B) X-Z reconstructions from confocal fluorescence imaging of intact, fully polarized monolayers of MDCK cells transfected with the human TfnR. Cells were loaded with FITC-Tfn for 20 min at 37°C (top). The cells were chased in the presence of unlabeled Tfn for 40 min in the absence (middle) or presence (bottom) of AlF 4 . Arrows indicate the apically located FITC-Tfn–containing endosomes; gray lines indicate the position of the Transwell filters and thus the basolateral surface of the cell monolayer. Each panel has been contrast processed with Adobe Photoshop to emphasize the position of the label rather than the total amount. (C) Optiprep gradient centrifugation of MDCK cells after 125 I-Tfn uptake for 2.5 min (squares) or 25 min (circles) in the presence (closed symbols) or absence (open symbols) of AlF 4 . Low density fractions are shown to the left.

    Journal: The Journal of Cell Biology

    Article Title: The Receptor Recycling Pathway Contains Two Distinct Populations of Early Endosomes with Different Sorting Functions

    doi:

    Figure Lengend Snippet: Partial retention of FITC-Tfn in REs by AlF 4 . (A) CHO cells and MDCK cells expressing human TfnR were pulsed with FITC-Tfn for 30 min at 37°C, conditions which labeled both peripheral and perinuclear endosome populations (panels A and B). The cells were then chased in the presence of excess unlabeled Tfn for 30 min (panels C and D). Although a few cells remained brightly labeled, most exhibited a marked loss of FITC-Tfn, with remaining cell-associated FITC-Tfn found predominantly in perinuclear vesicles (arrows). When cells were chased in the presence of unlabeled Tfn and AlF 4 for 30 min, however, FITC-Tfn was largely retained in the perinuclear endosomes (panels E and F). In CHO cells, these structures exhibited the characteristic distribution as a tightly organized array near the microtubule organizing center; in MDCK cells, they were more diffusely distributed, but nevertheless closely apposed to the nucleus. (B) X-Z reconstructions from confocal fluorescence imaging of intact, fully polarized monolayers of MDCK cells transfected with the human TfnR. Cells were loaded with FITC-Tfn for 20 min at 37°C (top). The cells were chased in the presence of unlabeled Tfn for 40 min in the absence (middle) or presence (bottom) of AlF 4 . Arrows indicate the apically located FITC-Tfn–containing endosomes; gray lines indicate the position of the Transwell filters and thus the basolateral surface of the cell monolayer. Each panel has been contrast processed with Adobe Photoshop to emphasize the position of the label rather than the total amount. (C) Optiprep gradient centrifugation of MDCK cells after 125 I-Tfn uptake for 2.5 min (squares) or 25 min (circles) in the presence (closed symbols) or absence (open symbols) of AlF 4 . Low density fractions are shown to the left.

    Article Snippet: Western Blot Fractions from Optiprep density gradients were mixed with an equal volume of H2 O containing 2% (wt/vol) sodium deoxycholate ( Sigma Chemical Co. ) and vortexed.

    Techniques: Expressing, Labeling, Fluorescence, Imaging, Transfection, Gradient Centrifugation

    Atg9 vesicles are generated at the Rab11-positive compartments upon nutrient starvation ( A ) Post-nuclear cell homogenates prepared from HeLa/S cells were subjected to subcellular fractionation using a continuous 5∼40% OptiPrep gradient and analyzed by immunoblotting using the indicated antibodies. More than 80% of Atg9 proteins in the homogenates were fractionated in the Rab11-positive fractions (Fractions #7∼9). ( B ) HeLa/Atg9-GFP cells were incubated in complete medium or starved in the presence or absence of 80 μM Dynasore for 1.5 h, stained for Rab11 and analyzed by confocal microscopy. Magnified images in the boxed areas are shown in the bottom. 3D SR, a 3D surface rendered image of the boxed area. ( C ) HeLa/Atg9-GFP cells were nucleofected with DsRed-Rab11 for 24 h. Cells were incubated in starvation medium for 45 min and then Dynasore (80 μM) was added in the medium. 3D time-lapse images were captured at 30-sec intervals beginning 10 min after the addition of Dynasore. 3D projection images at 0 and 20 min time points are shown on the left. Magnified images in the boxed area at the indicated time points are shown in the right. Arrows indicate an Atg9 and Rab11-positive compartment that underwent tubulation upon treatment. Scale bars represent 10 μm, and 2 μm in the magnified images.

    Journal: Oncotarget

    Article Title: The Bif-1-Dynamin 2 membrane fission machinery regulates Atg9-containing vesicle generation at the Rab11-positive reservoirs

    doi: 10.18632/oncotarget.8028

    Figure Lengend Snippet: Atg9 vesicles are generated at the Rab11-positive compartments upon nutrient starvation ( A ) Post-nuclear cell homogenates prepared from HeLa/S cells were subjected to subcellular fractionation using a continuous 5∼40% OptiPrep gradient and analyzed by immunoblotting using the indicated antibodies. More than 80% of Atg9 proteins in the homogenates were fractionated in the Rab11-positive fractions (Fractions #7∼9). ( B ) HeLa/Atg9-GFP cells were incubated in complete medium or starved in the presence or absence of 80 μM Dynasore for 1.5 h, stained for Rab11 and analyzed by confocal microscopy. Magnified images in the boxed areas are shown in the bottom. 3D SR, a 3D surface rendered image of the boxed area. ( C ) HeLa/Atg9-GFP cells were nucleofected with DsRed-Rab11 for 24 h. Cells were incubated in starvation medium for 45 min and then Dynasore (80 μM) was added in the medium. 3D time-lapse images were captured at 30-sec intervals beginning 10 min after the addition of Dynasore. 3D projection images at 0 and 20 min time points are shown on the left. Magnified images in the boxed area at the indicated time points are shown in the right. Arrows indicate an Atg9 and Rab11-positive compartment that underwent tubulation upon treatment. Scale bars represent 10 μm, and 2 μm in the magnified images.

    Article Snippet: The resultant post-nuclear cell homogenates (0.8 ml) were loaded on 6.4 ml of a 5–40% continuous OptiPrep (SIGMA, D1556) gradient and centrifuged at 36,000 rpm for 20 h at 4°C using a SW-41 swing rotor.

    Techniques: Generated, Fractionation, Incubation, Staining, Confocal Microscopy, Size-exclusion Chromatography

    Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

    Journal: Scientific Reports

    Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

    doi: 10.1038/s41598-017-09972-x

    Figure Lengend Snippet: Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

    Article Snippet: Endoplasmic reticulum purification ER Purification was carried out as previously described (Liao, HJ and Carpenter, G. 2007) with minor modifications, by using the OptiPrep density gradient system (Sigma-Aldrich, St Louis, Missouri, USA).

    Techniques: Purification, Marker, Western Blot, Isolation, SDS Page