Structured Review

PerkinElmer gamma counter
Blood clearance of the 99m Tc-RGD4CβL. Three Wistar rats were injected with the 99m Tc-RGD4CβL. Blood was drawn at different time-points, and radioactivities were measured by a <t>gamma</t> counter, data are shown as %ID/g, T 1/2 α and T 1/2 β were 7.8 and 21.9 min respectively.
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Images

1) Product Images from "A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy"

Article Title: A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms16059625

Blood clearance of the 99m Tc-RGD4CβL. Three Wistar rats were injected with the 99m Tc-RGD4CβL. Blood was drawn at different time-points, and radioactivities were measured by a gamma counter, data are shown as %ID/g, T 1/2 α and T 1/2 β were 7.8 and 21.9 min respectively.
Figure Legend Snippet: Blood clearance of the 99m Tc-RGD4CβL. Three Wistar rats were injected with the 99m Tc-RGD4CβL. Blood was drawn at different time-points, and radioactivities were measured by a gamma counter, data are shown as %ID/g, T 1/2 α and T 1/2 β were 7.8 and 21.9 min respectively.

Techniques Used: Injection

2) Product Images from "Performance assessment of a NaI(Tl) gamma counter for PET applications with methods for improved quantitative accuracy and greater standardization"

Article Title: Performance assessment of a NaI(Tl) gamma counter for PET applications with methods for improved quantitative accuracy and greater standardization

Journal: EJNMMI physics

doi: 10.1186/s40658-015-0114-3

Deadtime-corrected gamma counter CPM data as a function of activity in a decaying 18 F sample. The solid line indicates a linear fit to low count rate data and represents an idealized response under the assumption of perfect deadtime correction.
Figure Legend Snippet: Deadtime-corrected gamma counter CPM data as a function of activity in a decaying 18 F sample. The solid line indicates a linear fit to low count rate data and represents an idealized response under the assumption of perfect deadtime correction.

Techniques Used: Activity Assay

18 F energy spectra measured with the 2480 Wizard 2 gamma counter. When the source was located in the conventional position, inside the well (b) , a peak at 511 keV and a coincidence sum peak at 1,022 keV were seen (a) . When the source was positioned (for illustrative purposes) just outside the well (d) , it was not possible for corresponding annihilation photons to be simultaneously measured and, as a result, the 1,022-keV coincidence sum peak was absent from the spectrum (c) . Note that the photographs were taken with the shielding removed to show the two different positions of the (white) source holder.
Figure Legend Snippet: 18 F energy spectra measured with the 2480 Wizard 2 gamma counter. When the source was located in the conventional position, inside the well (b) , a peak at 511 keV and a coincidence sum peak at 1,022 keV were seen (a) . When the source was positioned (for illustrative purposes) just outside the well (d) , it was not possible for corresponding annihilation photons to be simultaneously measured and, as a result, the 1,022-keV coincidence sum peak was absent from the spectrum (c) . Note that the photographs were taken with the shielding removed to show the two different positions of the (white) source holder.

Techniques Used:

3) Product Images from "Interleukin 12-mediated Prevention of Spontaneous Mammary Adenocarcinomas in Two Lines of Her-2/neu Transgenic Mice "

Article Title: Interleukin 12-mediated Prevention of Spontaneous Mammary Adenocarcinomas in Two Lines of Her-2/neu Transgenic Mice

Journal: The Journal of Experimental Medicine

doi:

Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and IFN-γ ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.
Figure Legend Snippet: Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and IFN-γ ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.

Techniques Used: Mouse Assay, Staining

4) Product Images from "Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer"

Article Title: Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

Journal: Gene Therapy

doi: 10.1038/gt.2016.5

In vivo GLV-1h153 gene expression and therapy. ( a ) Gamma emissions from excised PC3 xenografts treated with intratumoural injection of 1 × 10 6 PFU GLV-1h153 and 1 mCi of 131I 48 h later, alongside controls that received 131 I only. ( b ) Viral GFP expression in intratumourally treated xenografts. ( c ) Long-term therapeutic effect of treatment with GLV-1h153 and 131 I on PC3 xenografts. Kaplan–Meier plot significance is the result of log-rank (Mantel Cox) test. * P
Figure Legend Snippet: In vivo GLV-1h153 gene expression and therapy. ( a ) Gamma emissions from excised PC3 xenografts treated with intratumoural injection of 1 × 10 6 PFU GLV-1h153 and 1 mCi of 131I 48 h later, alongside controls that received 131 I only. ( b ) Viral GFP expression in intratumourally treated xenografts. ( c ) Long-term therapeutic effect of treatment with GLV-1h153 and 131 I on PC3 xenografts. Kaplan–Meier plot significance is the result of log-rank (Mantel Cox) test. * P

Techniques Used: In Vivo, Expressing, Injection

5) Product Images from "Resveratrol Inhibits Sodium/Iodide Symporter Gene Expression and Function in Rat Thyroid Cells"

Article Title: Resveratrol Inhibits Sodium/Iodide Symporter Gene Expression and Function in Rat Thyroid Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107936

Effects of resveratrol on radioiodine uptake by the thyroid gland in vivo . Male Sprague-Dawley rats were treated with the control vehicle (Control, n = 6) or with 50 mg/kg/day resveratrol i.p. (Resv, n = 6), for 14 days. On the last day of treatment, the animals received [ 125 I]-NaI (185 kBq i.p., each) 24 h prior to sacrifice. Their thyroids were removed and weighed, with their associated radioactivity (RAIU) determined in a gamma counter. Data (from iodide uptake per thyroid weight) are normalized means ±SD (control vehicle, 100%). *, p
Figure Legend Snippet: Effects of resveratrol on radioiodine uptake by the thyroid gland in vivo . Male Sprague-Dawley rats were treated with the control vehicle (Control, n = 6) or with 50 mg/kg/day resveratrol i.p. (Resv, n = 6), for 14 days. On the last day of treatment, the animals received [ 125 I]-NaI (185 kBq i.p., each) 24 h prior to sacrifice. Their thyroids were removed and weighed, with their associated radioactivity (RAIU) determined in a gamma counter. Data (from iodide uptake per thyroid weight) are normalized means ±SD (control vehicle, 100%). *, p

Techniques Used: In Vivo, Radioactivity

6) Product Images from "Docetaxel-titanate nanotubes enhance radiosensitivity in an androgen-independent prostate cancer model"

Article Title: Docetaxel-titanate nanotubes enhance radiosensitivity in an androgen-independent prostate cancer model

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S139167

TiONts-DTX biodistribution analysis. Notes: ( A ) SPECT-CT imaging of kinetics and biodistribution analysis for each organ (expressed as a percentage of injected 111 In activity, taking into account the decrease in 111 In activity after the injection of DOTA[ 111 In] ( A 1) or TiONts-DTX-DOTA[ 111 In] ( A 2)). ( B ) TiONts-DTX-DOTA[ 111 In] biodistribution in dissected organs by radioactivity detection using gamma counting 7 days after injection (mean value ± SD). ( C ) TEM images showing the intracellular location of TiONts-DTX 24 h after injection into PC-3 tumors. Abbreviations: DTX, docetaxel; SPECT-CT, single-photon emission computed tomography-computed tomography; TEM, transmission electron microscopy; TiONts, titanate nanotubes.
Figure Legend Snippet: TiONts-DTX biodistribution analysis. Notes: ( A ) SPECT-CT imaging of kinetics and biodistribution analysis for each organ (expressed as a percentage of injected 111 In activity, taking into account the decrease in 111 In activity after the injection of DOTA[ 111 In] ( A 1) or TiONts-DTX-DOTA[ 111 In] ( A 2)). ( B ) TiONts-DTX-DOTA[ 111 In] biodistribution in dissected organs by radioactivity detection using gamma counting 7 days after injection (mean value ± SD). ( C ) TEM images showing the intracellular location of TiONts-DTX 24 h after injection into PC-3 tumors. Abbreviations: DTX, docetaxel; SPECT-CT, single-photon emission computed tomography-computed tomography; TEM, transmission electron microscopy; TiONts, titanate nanotubes.

Techniques Used: Single Photon Emission Computed Tomography, Imaging, Injection, Activity Assay, Radioactivity, Transmission Electron Microscopy, Computed Tomography, Transmission Assay, Electron Microscopy

7) Product Images from "Anti PD-1 treatment increases [18F]FDG uptake by cancer cells in a mouse B16F10 melanoma model"

Article Title: Anti PD-1 treatment increases [18F]FDG uptake by cancer cells in a mouse B16F10 melanoma model

Journal: EJNMMI Research

doi: 10.1186/s13550-018-0433-1

Ex vivo validation of [ 18 F]FDG uptake. a Evaluated the percentage-injected activity per gram of tissue (%IA/g) by gamma counter in tumors, spleens and blood on day 7 ( n = 7). b Representative HE-stained and autoradiography (ARG) images of treatment group tumor (top) or non-treatment group tumor (bottom). Data represent mean ± SEM; * P
Figure Legend Snippet: Ex vivo validation of [ 18 F]FDG uptake. a Evaluated the percentage-injected activity per gram of tissue (%IA/g) by gamma counter in tumors, spleens and blood on day 7 ( n = 7). b Representative HE-stained and autoradiography (ARG) images of treatment group tumor (top) or non-treatment group tumor (bottom). Data represent mean ± SEM; * P

Techniques Used: Ex Vivo, Injection, Activity Assay, IA, Staining, Autoradiography

8) Product Images from "VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis"

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

Journal: Scientific Reports

doi: 10.1038/s41598-018-21491-x

Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ) NIH3T3 cells transfected with the indicated plasmids were harvested 24 h post transfection and subjected to western blot analysis using anti-γ-tubulin antibodies. Equal total protein amounts were loaded (see also supplementary Fig. S9 ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ) (see also supplementary Fig. S9 ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.
Figure Legend Snippet: Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ) NIH3T3 cells transfected with the indicated plasmids were harvested 24 h post transfection and subjected to western blot analysis using anti-γ-tubulin antibodies. Equal total protein amounts were loaded (see also supplementary Fig. S9 ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ) (see also supplementary Fig. S9 ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

Techniques Used: Staining, Transfection, Construct, Cotransfection, Western Blot

ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ) Percentage of cells exhibiting centrosome localization of the indicated proteins (corresponds to Fig. 2a and supplementary Fig. S2a ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.
Figure Legend Snippet: ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ) Percentage of cells exhibiting centrosome localization of the indicated proteins (corresponds to Fig. 2a and supplementary Fig. S2a ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

Techniques Used: Transfection, Expressing, Microscopy, Cotransfection

A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.
Figure Legend Snippet: A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

Techniques Used: Inhibition, Mutagenesis

Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ . Cells were then processed for electron microscopy as described in material and methods. Cells selected by fluorescence microscopy were located in the TEM and serial sections were collected to locate the centrosome (supplementary Fig. S3 ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.
Figure Legend Snippet: Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ . Cells were then processed for electron microscopy as described in material and methods. Cells selected by fluorescence microscopy were located in the TEM and serial sections were collected to locate the centrosome (supplementary Fig. S3 ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

Techniques Used: Expressing, Staining, Electron Microscopy, Fluorescence, Microscopy, Transmission Electron Microscopy

Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ . Shown are time composite of sequential frames acquired for EB1-GFP channel on a spinning-disk confocal microscope at 1 second intervals for 1.5 minutes from representative cells (entire movie series are provided in supplementary Movies 1 and 2 ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.
Figure Legend Snippet: Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ . Shown are time composite of sequential frames acquired for EB1-GFP channel on a spinning-disk confocal microscope at 1 second intervals for 1.5 minutes from representative cells (entire movie series are provided in supplementary Movies 1 and 2 ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

Techniques Used: Activity Assay, Transfection, Binding Assay, Microscopy, Expressing, Marker, Staining

9) Product Images from "Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice"

Article Title: Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice

Journal: Scientific Reports

doi: 10.1038/s41598-018-29128-9

Stx2a is actively released into the culture medium depending on its B-subunit. ( A ) Vero cells were treated with 1 μg/ml of 125 I- Stx1a or 125 I- Stx2a for 2 hr at 37 °C, and then further cultured for 1 hr without Stx to incorporate the toxin. After washing, the cells were cultured for the indicated period. At each time point, cell lysates and TCA-ppt of the culture medium were prepared and then separated by electrophoresis and visualized (whole images are presented in Supplementary Fig. S1 ). Radioactivity of the TCA-sup, the TCA-ppt, and the cell lysates was measured by a γ-counter. The data are presented as the percentage of the total radioactivity (mean ± SE, n = 3). ( B ) Purified Stx1a (1 μg), Stx2a (1 μg), Stx1A2B, and Stx2A1B were analyzed by electrophoresis on an SDS/16% PAGE and visualized by CBB staining. ( C ) Vero cells were treated with 1 μg/ml of 125 I-Stx1A2B or 125 I- Stx2A1B for 2 hr at 37 °C and then further cultured another 1 hr without Stx. The metabolism of each Stx was analyzed as described in ( A ). Whole images are presented in Supplementary Fig. S2 . The data are presented as the percentage of the total radioactivity (mean ± SE, n = 3).
Figure Legend Snippet: Stx2a is actively released into the culture medium depending on its B-subunit. ( A ) Vero cells were treated with 1 μg/ml of 125 I- Stx1a or 125 I- Stx2a for 2 hr at 37 °C, and then further cultured for 1 hr without Stx to incorporate the toxin. After washing, the cells were cultured for the indicated period. At each time point, cell lysates and TCA-ppt of the culture medium were prepared and then separated by electrophoresis and visualized (whole images are presented in Supplementary Fig. S1 ). Radioactivity of the TCA-sup, the TCA-ppt, and the cell lysates was measured by a γ-counter. The data are presented as the percentage of the total radioactivity (mean ± SE, n = 3). ( B ) Purified Stx1a (1 μg), Stx2a (1 μg), Stx1A2B, and Stx2A1B were analyzed by electrophoresis on an SDS/16% PAGE and visualized by CBB staining. ( C ) Vero cells were treated with 1 μg/ml of 125 I-Stx1A2B or 125 I- Stx2A1B for 2 hr at 37 °C and then further cultured another 1 hr without Stx. The metabolism of each Stx was analyzed as described in ( A ). Whole images are presented in Supplementary Fig. S2 . The data are presented as the percentage of the total radioactivity (mean ± SE, n = 3).

Techniques Used: Cell Culture, Electrophoresis, Radioactivity, Purification, Polyacrylamide Gel Electrophoresis, Staining

10) Product Images from "Neutralization of membrane complement regulators improves complement-dependent effector functions of therapeutic anticancer antibodies targeting leukemic cells"

Article Title: Neutralization of membrane complement regulators improves complement-dependent effector functions of therapeutic anticancer antibodies targeting leukemic cells

Journal: Oncoimmunology

doi: 10.4161/2162402X.2014.979688

CDCC and macrophage mediated phagocytosis of leukemic cells upon mCRP neutralization. CDCC was analyzed by 51 Cr release assay. Raji and REH cells were transfected with control siRNA or combined siRNA anti-CD46, CD55, and CD59 or left untreated. 72 h after siRNA transfection, Raji cells were incubated with ( A ) Rituximab (RTX; 10 μg/mL) ( B ) REH cells with Alemtuzumab (ALM; 10 μg/mL) ( C ) Three CLL samples were incubated either with RTX or OFA or ALM (each 10 μg/mL) alone or in combination with anti-CD46 and anti-CD55 neutralizing antibodies, followed by the addition of C8 depleted serum or heat-inactivated serum. After 30 min, in vitro differentiated macrophages (E:T – 10:1) were added to tumor cells and incubated for 4 h. Radioactivity in supernatants was measured in a γ-counter. Data are given as mean values ± SD of n = 3; p
Figure Legend Snippet: CDCC and macrophage mediated phagocytosis of leukemic cells upon mCRP neutralization. CDCC was analyzed by 51 Cr release assay. Raji and REH cells were transfected with control siRNA or combined siRNA anti-CD46, CD55, and CD59 or left untreated. 72 h after siRNA transfection, Raji cells were incubated with ( A ) Rituximab (RTX; 10 μg/mL) ( B ) REH cells with Alemtuzumab (ALM; 10 μg/mL) ( C ) Three CLL samples were incubated either with RTX or OFA or ALM (each 10 μg/mL) alone or in combination with anti-CD46 and anti-CD55 neutralizing antibodies, followed by the addition of C8 depleted serum or heat-inactivated serum. After 30 min, in vitro differentiated macrophages (E:T – 10:1) were added to tumor cells and incubated for 4 h. Radioactivity in supernatants was measured in a γ-counter. Data are given as mean values ± SD of n = 3; p

Techniques Used: Neutralization, Release Assay, Transfection, Incubation, In Vitro, Radioactivity

11) Product Images from "Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B"

Article Title: Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.v12.i27.4310

Addition of neutralizing anti-IL-10 antibody restores HBcAg-specific production of IFN-γ by CD4 + in patients with hepatitis B. PBMCs obtained from 5 patients with CHB and 7 healthy subjects were stimulated with HBcAg (10 μg/mL) for 9 h and thereafter cells were stained for IFN-γ-secretion (PE) and anti-CD4-PerCP to determine the population of HBcAg-specific T H 1 being identified as IFN-γ + cells in CD4 + T cells (A). Anti-IL-10 neutralizing antibody or isotype-matched control antibody were added to the culture during stimulation with HBcAg. The addition of anti-IL-10 antibody did not affect the percentage of CD4 + T cells (B). In culture with anti-IL-10 antibody, numbers of HBcAg-specific T H 1 were significantly higher than those in culture with a control antibody (C).
Figure Legend Snippet: Addition of neutralizing anti-IL-10 antibody restores HBcAg-specific production of IFN-γ by CD4 + in patients with hepatitis B. PBMCs obtained from 5 patients with CHB and 7 healthy subjects were stimulated with HBcAg (10 μg/mL) for 9 h and thereafter cells were stained for IFN-γ-secretion (PE) and anti-CD4-PerCP to determine the population of HBcAg-specific T H 1 being identified as IFN-γ + cells in CD4 + T cells (A). Anti-IL-10 neutralizing antibody or isotype-matched control antibody were added to the culture during stimulation with HBcAg. The addition of anti-IL-10 antibody did not affect the percentage of CD4 + T cells (B). In culture with anti-IL-10 antibody, numbers of HBcAg-specific T H 1 were significantly higher than those in culture with a control antibody (C).

Techniques Used: Staining

Depletion of CD4 + CD25 + T cells from PBMCs increases HBcAg-specific production of IFN-γ in patients with hepatitis B. Using the differential expression of CD4 and CD25, cells were separated into 3 fractions; fraction 1 consisted of CD4 - cells, fraction 2 consisted of CD4 + CD25 - cells and fraction 3 consisted of CD4 + CD25 + cells (A). Thereafter, 2 sets of lymphocyte preparations were reconstituted by remixing fractions 1, 2 and 3 or by remixing fractions 1 and 2 (B). They were stimulated with HBcAg to finally stain a CD4 + IFN-γ + population (C).
Figure Legend Snippet: Depletion of CD4 + CD25 + T cells from PBMCs increases HBcAg-specific production of IFN-γ in patients with hepatitis B. Using the differential expression of CD4 and CD25, cells were separated into 3 fractions; fraction 1 consisted of CD4 - cells, fraction 2 consisted of CD4 + CD25 - cells and fraction 3 consisted of CD4 + CD25 + cells (A). Thereafter, 2 sets of lymphocyte preparations were reconstituted by remixing fractions 1, 2 and 3 or by remixing fractions 1 and 2 (B). They were stimulated with HBcAg to finally stain a CD4 + IFN-γ + population (C).

Techniques Used: Expressing, Staining

Comparison of levels of mRNAs for T-bet and GATA-3 after stimulation with HBsAg and HBcAg with mRNAs for IFN-gamma, IL-10 and IL-4. Total cellular RNA was extracted from CD4 + T cells after the stimulation of PBMCs with HBcAg (10 μg/mL) or HBsAg (29 μg/mL) for 24 h. A: HBcAg stimulation; B: HBsAg stimulation. Levels of mRNA for T-bet, GATA-3, IFN-γ, IL-12R β2 and IL-4 were quantified by TaqMan PCR. GAPDH was used as an internal control. Relative amount of target mRNA was calculated using comparative CT method. The expression level of mRNAs of the non-stimulated sample in each subject is represented as 1.0 and relative amount of target mRNA in a stimulated sample was calculated using the as following formula: relative amount = 2 -ΔΔ C T , where ΔΔC T was given by subtracting ΔC T (non-stimulated cells) from ΔC T (stimulated cells). The ΔC T value was determined by subtracting the GAPDH C T value from the target C T value. The validation experiments were performed in advance for all the target mRNAs to demonstrate that efficiency of each target and GAPDH are approximately equal.
Figure Legend Snippet: Comparison of levels of mRNAs for T-bet and GATA-3 after stimulation with HBsAg and HBcAg with mRNAs for IFN-gamma, IL-10 and IL-4. Total cellular RNA was extracted from CD4 + T cells after the stimulation of PBMCs with HBcAg (10 μg/mL) or HBsAg (29 μg/mL) for 24 h. A: HBcAg stimulation; B: HBsAg stimulation. Levels of mRNA for T-bet, GATA-3, IFN-γ, IL-12R β2 and IL-4 were quantified by TaqMan PCR. GAPDH was used as an internal control. Relative amount of target mRNA was calculated using comparative CT method. The expression level of mRNAs of the non-stimulated sample in each subject is represented as 1.0 and relative amount of target mRNA in a stimulated sample was calculated using the as following formula: relative amount = 2 -ΔΔ C T , where ΔΔC T was given by subtracting ΔC T (non-stimulated cells) from ΔC T (stimulated cells). The ΔC T value was determined by subtracting the GAPDH C T value from the target C T value. The validation experiments were performed in advance for all the target mRNAs to demonstrate that efficiency of each target and GAPDH are approximately equal.

Techniques Used: Polymerase Chain Reaction, Expressing

12) Product Images from "Evaluation of an [18F]AlF-NOTA Analog of Exendin-4 for Imaging of GLP-1 Receptor in Insulinoma"

Article Title: Evaluation of an [18F]AlF-NOTA Analog of Exendin-4 for Imaging of GLP-1 Receptor in Insulinoma

Journal: Theranostics

doi: 10.7150/thno.5276

A) Representative PET images of [ 18 F]AlF-NOTA-MAL-cys 40 -exendin-4 in INS-1 xenografted mice at 30 and 60 min p.i.. Tumor uptake is completely blocked by co-administration of a blocking dose of exendin-4. B) Quantification of uptake by selected tissues determined by ROIs following injection of [ 18 F]AlF-NOTA-MAL-cys 40 -exendin-4 or co-injection of blocking agent (exendin-4) into INS-1 tumor-bearing mice at 30 and 60 min p.i.. C) Experiment of B at 60 min with analysis by tissue dissection and gamma counting.
Figure Legend Snippet: A) Representative PET images of [ 18 F]AlF-NOTA-MAL-cys 40 -exendin-4 in INS-1 xenografted mice at 30 and 60 min p.i.. Tumor uptake is completely blocked by co-administration of a blocking dose of exendin-4. B) Quantification of uptake by selected tissues determined by ROIs following injection of [ 18 F]AlF-NOTA-MAL-cys 40 -exendin-4 or co-injection of blocking agent (exendin-4) into INS-1 tumor-bearing mice at 30 and 60 min p.i.. C) Experiment of B at 60 min with analysis by tissue dissection and gamma counting.

Techniques Used: Positron Emission Tomography, Mouse Assay, Blocking Assay, Injection, Dissection

13) Product Images from "Interferon-γ PET Imaging as a Predictive Tool for Monitoring Response to Tumor Immunotherapy"

Article Title: Interferon-γ PET Imaging as a Predictive Tool for Monitoring Response to Tumor Immunotherapy

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-18-0253

Validation of specificity of 89 Zr-anti-IFN-γ. A) BALB/c mice treated with CpG-ODN and imaged with the tracer 72 h p.i. displayed higher uptake in the spleen compared to control (Ctrl) untreated cohorts (n=3 each). B) Tissue distribution of 89 Zr-anti-IFN-γ at 72 h p.i. demonstrated lower probe accumulation in the spleen upon competitive saturation with 10× cold AN-18 mAb (n=4 each). C) Binding of 89 Zr-anti-IFN-γ receptor-localized IFN-γ was tested in vitro . TUBO cells were incubated with 89 Zr-anti-IFN-γ alone (n=5), or with recombinant IFN-γ (rIFN-γ) and washed before addition of 89 Zr-anti-IFN-γ (n=5). Activity was measured by a gamma counter and adjusted for cell count.
Figure Legend Snippet: Validation of specificity of 89 Zr-anti-IFN-γ. A) BALB/c mice treated with CpG-ODN and imaged with the tracer 72 h p.i. displayed higher uptake in the spleen compared to control (Ctrl) untreated cohorts (n=3 each). B) Tissue distribution of 89 Zr-anti-IFN-γ at 72 h p.i. demonstrated lower probe accumulation in the spleen upon competitive saturation with 10× cold AN-18 mAb (n=4 each). C) Binding of 89 Zr-anti-IFN-γ receptor-localized IFN-γ was tested in vitro . TUBO cells were incubated with 89 Zr-anti-IFN-γ alone (n=5), or with recombinant IFN-γ (rIFN-γ) and washed before addition of 89 Zr-anti-IFN-γ (n=5). Activity was measured by a gamma counter and adjusted for cell count.

Techniques Used: Mouse Assay, Binding Assay, In Vitro, Incubation, Recombinant, Activity Assay, Cell Counting

14) Product Images from "Arthritogenicity of collagen type II is increased by chlorination"

Article Title: Arthritogenicity of collagen type II is increased by chlorination

Journal: Clinical and Experimental Immunology

doi: 10.1111/j.1365-2249.2006.03129.x

Analysis of interferon (IFN)-γ and interkeukin (IL)-1β mRNA expression in draining lymph nodes 7 days post-immunization (p.i.). Levels of mRNA are expressed as ratios of GADPH mRNA levels. (a) IFN-γ mRNA was expressed to significantly higher levels in collagen type II (CII)–Cl-immunized rats compared to CII-immunized rats. P -value 0·047 calculated by Mann–Whitney test for nonparametric samples. (b) IL-1β mRNA levels were fourfold higher in rats immunized with CII–Cl compared with rats immunized with CII. A P - value of 0·009 was obtained as calculated by Mann–Whitney test for nonparametric samples. The analysis was performed on five rats in each group and repeated twice.
Figure Legend Snippet: Analysis of interferon (IFN)-γ and interkeukin (IL)-1β mRNA expression in draining lymph nodes 7 days post-immunization (p.i.). Levels of mRNA are expressed as ratios of GADPH mRNA levels. (a) IFN-γ mRNA was expressed to significantly higher levels in collagen type II (CII)–Cl-immunized rats compared to CII-immunized rats. P -value 0·047 calculated by Mann–Whitney test for nonparametric samples. (b) IL-1β mRNA levels were fourfold higher in rats immunized with CII–Cl compared with rats immunized with CII. A P - value of 0·009 was obtained as calculated by Mann–Whitney test for nonparametric samples. The analysis was performed on five rats in each group and repeated twice.

Techniques Used: Expressing, MANN-WHITNEY

15) Product Images from "CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression"

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku263

Suv39H1 can form complex with CDK2 but not with CDK1 in vivo. ( A ) Potential phosphorylation sites of human Suv39H1. Putative phosphorylation sites are shown in black and the phosphorylation site with the CDK1 or CDK2 consensus motif (S391 residue) is shown in red. ( B and C ) Suv39H1 interacts with CDK2 but not with CDK1 in vivo . Total cell lysates from 293T cells co-transfected with Myc-Suv39H1 and Flag-CDK1 or Flag-CDK2 (in panel (B)) or Flag-Suv39H1 and Myc-CDK1 or Myc-CDK2 (in panel (C)) were co-immunoprecipitated (IP) using anti-Myc agarose beads. The IP fractions were analyzed by western blotting using anti-Myc or anti-Flag antibody. The expression of each gene in whole cell lysate (WCL) was confirmed by western blot analysis with anti-Flag or anti-Myc antibodies. ( D ) In vitro kinase assay with purified MBP-fused Suv39H1-WT or MBP-Suv39H1-S391A mutant protein. Purified MBP-Suv39H1-WT and MBP-Suv39H1-S391A mutant proteins were incubated with gamma- 32 P-ATP and CDK2/cyclin complexes. The phosphorylation was visualized by exposure to X-ray film. The data showed that MBP-Suv39H1-WT was phosphorylated by both CDK2/cyclin A2 and CDK2/cyclin E1 complexes but not MBP-Suv39H1-S391A mutant.
Figure Legend Snippet: Suv39H1 can form complex with CDK2 but not with CDK1 in vivo. ( A ) Potential phosphorylation sites of human Suv39H1. Putative phosphorylation sites are shown in black and the phosphorylation site with the CDK1 or CDK2 consensus motif (S391 residue) is shown in red. ( B and C ) Suv39H1 interacts with CDK2 but not with CDK1 in vivo . Total cell lysates from 293T cells co-transfected with Myc-Suv39H1 and Flag-CDK1 or Flag-CDK2 (in panel (B)) or Flag-Suv39H1 and Myc-CDK1 or Myc-CDK2 (in panel (C)) were co-immunoprecipitated (IP) using anti-Myc agarose beads. The IP fractions were analyzed by western blotting using anti-Myc or anti-Flag antibody. The expression of each gene in whole cell lysate (WCL) was confirmed by western blot analysis with anti-Flag or anti-Myc antibodies. ( D ) In vitro kinase assay with purified MBP-fused Suv39H1-WT or MBP-Suv39H1-S391A mutant protein. Purified MBP-Suv39H1-WT and MBP-Suv39H1-S391A mutant proteins were incubated with gamma- 32 P-ATP and CDK2/cyclin complexes. The phosphorylation was visualized by exposure to X-ray film. The data showed that MBP-Suv39H1-WT was phosphorylated by both CDK2/cyclin A2 and CDK2/cyclin E1 complexes but not MBP-Suv39H1-S391A mutant.

Techniques Used: In Vivo, Transfection, Immunoprecipitation, Western Blot, Expressing, In Vitro, Kinase Assay, Purification, Mutagenesis, Incubation

16) Product Images from "Disruption of TGF-? signaling in T cells accelerates atherosclerosis"

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200318607

Serum anti–MDA-LDL Ab’s and cytokines in mice with ApoE deficiency and/or abrogated TGF-β signaling in T cells. ( a ) Titers of IgG Ab’s to MDA-LDL in 24-week-old male mice carrying intact or targeted ApoE genes, dominant negative CD4dnTβRII genes, or both. OD values of ELISA, dot plots with values for individual mice, and medians per group. ( b – e ) Cytokine concentrations of IFN-γ, TNF-α, IL-5, and IL-2 in sera from mice with intact or targeted ApoE genes, dominant negative CD4dnTβRII, or both, as determined by cytofluorometric bead assay. Dot plots as above.
Figure Legend Snippet: Serum anti–MDA-LDL Ab’s and cytokines in mice with ApoE deficiency and/or abrogated TGF-β signaling in T cells. ( a ) Titers of IgG Ab’s to MDA-LDL in 24-week-old male mice carrying intact or targeted ApoE genes, dominant negative CD4dnTβRII genes, or both. OD values of ELISA, dot plots with values for individual mice, and medians per group. ( b – e ) Cytokine concentrations of IFN-γ, TNF-α, IL-5, and IL-2 in sera from mice with intact or targeted ApoE genes, dominant negative CD4dnTβRII, or both, as determined by cytofluorometric bead assay. Dot plots as above.

Techniques Used: Multiple Displacement Amplification, Mouse Assay, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay

Effects of abrogated TGF-β signaling on T cell activation and cytokine secretion. ( a , b ) FACS analysis of the activation marker CD69 on CD4 + ( a ) and CD8 + ( b ) spleen T cells from 12-week-old E 0 × CD4dnTβRII ( n = 6, gray boxes) and E 0 mice ( n = 5, white boxes). Box plots (median, quartiles, tenth, and ninetieth percentiles) show percentage of CD69 + among all CD4 + or CD8 + T cells; staining for isotype control is subtracted. Experiments were repeated twice. ( c – f ) Cell proliferation and cytokine secretion after in vitro stimulation of spleen cells from 12-week-old E 0 × CD4dnT ( n = 6) and E 0 ( n = 5) mice. ( c ) Stimulation index after 3 H-thymidine incorporation. Concentrations of ( d ) IFN-γ, ( e ) IL-10, and ( f ) IL-4 in supernatants 48 hours after stimulation of spleen cells with the T cell mitogen, concavalin A. Box plots as above. ( g – j ) Concentrations of IFN-γ ( g ), TNF-α ( h ), IL-5 ( i ), and IL-2 ( j ) were determined in sera from 12-week-old E 0 × CD4dnTβRII and E 0 mice by using cytofluorometric bead assays. Dot plots show values for individual mice and medians for each group.
Figure Legend Snippet: Effects of abrogated TGF-β signaling on T cell activation and cytokine secretion. ( a , b ) FACS analysis of the activation marker CD69 on CD4 + ( a ) and CD8 + ( b ) spleen T cells from 12-week-old E 0 × CD4dnTβRII ( n = 6, gray boxes) and E 0 mice ( n = 5, white boxes). Box plots (median, quartiles, tenth, and ninetieth percentiles) show percentage of CD69 + among all CD4 + or CD8 + T cells; staining for isotype control is subtracted. Experiments were repeated twice. ( c – f ) Cell proliferation and cytokine secretion after in vitro stimulation of spleen cells from 12-week-old E 0 × CD4dnT ( n = 6) and E 0 ( n = 5) mice. ( c ) Stimulation index after 3 H-thymidine incorporation. Concentrations of ( d ) IFN-γ, ( e ) IL-10, and ( f ) IL-4 in supernatants 48 hours after stimulation of spleen cells with the T cell mitogen, concavalin A. Box plots as above. ( g – j ) Concentrations of IFN-γ ( g ), TNF-α ( h ), IL-5 ( i ), and IL-2 ( j ) were determined in sera from 12-week-old E 0 × CD4dnTβRII and E 0 mice by using cytofluorometric bead assays. Dot plots show values for individual mice and medians for each group.

Techniques Used: Activation Assay, FACS, Marker, Mouse Assay, Staining, In Vitro

Abrogation of TGF-β signaling in T cells increases atherosclerosis and IFN-γ expression in age-matched mice. ( a ) Lesion size in the aortic root (× 10 5 μm 2 ) in 12-week-old mice. Individual values are displayed by dots, and medians for each group are indicated by horizontal lines. Representative micrographs showing hematoxylin/oil red-O–stained lesions in E 0 × CD4dnTβRII ( b ) and E 0 ( c ) mice. Original magnification ×50. ( d ) Lipid lesion area in the entire aorta (percentage of total aortic surface area); dot plots as above. Sudan IV–stained en face preparations of aortas from E 0 × CD4dnTβRII ( e ) and E 0 ( f ) mice. ( g ) IFN-γ mRNA levels in lesions of E 0 × CD4dnTβRII and E 0 mice. Data are expressed as arbitrary units as described in Methods; dot plots as above.
Figure Legend Snippet: Abrogation of TGF-β signaling in T cells increases atherosclerosis and IFN-γ expression in age-matched mice. ( a ) Lesion size in the aortic root (× 10 5 μm 2 ) in 12-week-old mice. Individual values are displayed by dots, and medians for each group are indicated by horizontal lines. Representative micrographs showing hematoxylin/oil red-O–stained lesions in E 0 × CD4dnTβRII ( b ) and E 0 ( c ) mice. Original magnification ×50. ( d ) Lipid lesion area in the entire aorta (percentage of total aortic surface area); dot plots as above. Sudan IV–stained en face preparations of aortas from E 0 × CD4dnTβRII ( e ) and E 0 ( f ) mice. ( g ) IFN-γ mRNA levels in lesions of E 0 × CD4dnTβRII and E 0 mice. Data are expressed as arbitrary units as described in Methods; dot plots as above.

Techniques Used: Expressing, Mouse Assay, Staining

17) Product Images from "Functionalised Carbon Nanotubes Enhance Brain Delivery of Amyloid-Targeting Pittsburgh Compound B (PiB)-Derived Ligands"

Article Title: Functionalised Carbon Nanotubes Enhance Brain Delivery of Amyloid-Targeting Pittsburgh Compound B (PiB)-Derived Ligands

Journal: Nanotheranostics

doi: 10.7150/ntno.23125

Brain distribution profile of 111 In-labelled L 2 and L 3 , in its free form or conjugated to ox-MWNTs-NH 3 + , following systemic administration in healthy C57BL/6 mice. Mice were injected ( via tail vein) with 111 In-labelled L 2/3 or 111 In(L 2/3 ):ox-MWNTs-NH 3 + (50 μg, 0.5 MBq). After measuring the radioactivity of the whole organs, brain tissues were processed to separate brain parenchyma from capillaries ( via capillary depletion) and the radioactivity of each fraction was measured by gamma counting (A) , with the data presented as % ID per total brain parenchyma or capillaries. (B) For brain parenchyma to blood ratio calculation, the weight of parenchyma was considered as the weight of the whole brain. Inset: magnification of brain parenchyma/blood ratio for L 2 and L 3 . Statistical analysis of the results (mean ± SD, n = 3;) was performed using unpaired t-test with Welch's correction. (A) *** p
Figure Legend Snippet: Brain distribution profile of 111 In-labelled L 2 and L 3 , in its free form or conjugated to ox-MWNTs-NH 3 + , following systemic administration in healthy C57BL/6 mice. Mice were injected ( via tail vein) with 111 In-labelled L 2/3 or 111 In(L 2/3 ):ox-MWNTs-NH 3 + (50 μg, 0.5 MBq). After measuring the radioactivity of the whole organs, brain tissues were processed to separate brain parenchyma from capillaries ( via capillary depletion) and the radioactivity of each fraction was measured by gamma counting (A) , with the data presented as % ID per total brain parenchyma or capillaries. (B) For brain parenchyma to blood ratio calculation, the weight of parenchyma was considered as the weight of the whole brain. Inset: magnification of brain parenchyma/blood ratio for L 2 and L 3 . Statistical analysis of the results (mean ± SD, n = 3;) was performed using unpaired t-test with Welch's correction. (A) *** p

Techniques Used: Mouse Assay, Injection, Radioactivity

Blood and organ biodistribution profiles of 111 In-labelled L 2 and L 3 , in its free form or conjugated to ox-MWNTs-NH 3 + , following systemic administration in healthy C57BL/6 mice. Mice were injected ( via tail vein) with 111 In-labelled L 2/3 or 111 In(L 2/3 ):ox-MWNTs-NH 3 + (50 μg, 0.5 MBq). Blood was sampled at 2, 5, 10 and 30 min post-injection, followed by whole-body perfusion with heparinised saline. All major organs were immediately harvested and the radioactivity of (A) blood and (B) organs was measured by gamma counting. The Results are presented as (A) % injected dose per blood and (B) % injected dose per gram of tissue (inset: magnification of brain distribution). Statistical analysis of the results (mean ± SD, n = 3;) was performed using One-way ANOVA with Tukey's posthoc test. (A) *** p
Figure Legend Snippet: Blood and organ biodistribution profiles of 111 In-labelled L 2 and L 3 , in its free form or conjugated to ox-MWNTs-NH 3 + , following systemic administration in healthy C57BL/6 mice. Mice were injected ( via tail vein) with 111 In-labelled L 2/3 or 111 In(L 2/3 ):ox-MWNTs-NH 3 + (50 μg, 0.5 MBq). Blood was sampled at 2, 5, 10 and 30 min post-injection, followed by whole-body perfusion with heparinised saline. All major organs were immediately harvested and the radioactivity of (A) blood and (B) organs was measured by gamma counting. The Results are presented as (A) % injected dose per blood and (B) % injected dose per gram of tissue (inset: magnification of brain distribution). Statistical analysis of the results (mean ± SD, n = 3;) was performed using One-way ANOVA with Tukey's posthoc test. (A) *** p

Techniques Used: Mouse Assay, Injection, Radioactivity

18) Product Images from "Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain"

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain

Journal: Contrast media & molecular imaging

doi: 10.1002/cmmi.1701

Comparison of brain uptake of no-carrier-added (NCA) and carrier-added (CA) 52 Mn with ex vivo gamma counting and in vivo PET/CT. We observed higher activity uptake of NCA 52 Mn in both ex vivo gamma counting measurements of the excised brain at 24 and 48 hours (A, B) as well as in vivo with PET/CT for up to 7 days following contrast delivery (C). Ex vivo measurements are the average of 3 subjects per contrast agent formulation and time point, while in vivo PET/CT was performed on one subject per contrast agent formulation in this initial comparative imaging study.
Figure Legend Snippet: Comparison of brain uptake of no-carrier-added (NCA) and carrier-added (CA) 52 Mn with ex vivo gamma counting and in vivo PET/CT. We observed higher activity uptake of NCA 52 Mn in both ex vivo gamma counting measurements of the excised brain at 24 and 48 hours (A, B) as well as in vivo with PET/CT for up to 7 days following contrast delivery (C). Ex vivo measurements are the average of 3 subjects per contrast agent formulation and time point, while in vivo PET/CT was performed on one subject per contrast agent formulation in this initial comparative imaging study.

Techniques Used: Ex Vivo, In Vivo, Positron Emission Tomography, Activity Assay, Imaging

19) Product Images from "VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis"

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

Journal: Scientific Reports

doi: 10.1038/s41598-018-21491-x

Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.
Figure Legend Snippet: Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

Techniques Used: Staining, Transfection, Construct, Cotransfection, Western Blot

ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.
Figure Legend Snippet: ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

Techniques Used: Transfection, Expressing, Microscopy, Cotransfection

A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.
Figure Legend Snippet: A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

Techniques Used: Inhibition, Mutagenesis

Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.
Figure Legend Snippet: Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

Techniques Used: Expressing, Staining

Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.
Figure Legend Snippet: Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

Techniques Used: Activity Assay, Transfection, Binding Assay, Expressing, Marker, Staining, Microscopy

20) Product Images from "Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies"

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022257

Dynamic changes of NcoA4 association with the centrosome during mitotic progression. COS cells were stained for NcoA4 (red; B and E ), and γ-tubulin (green; A and D ) and examined under confocal microscopy. Merged images are shown in C and F and all images shown are of single optical slices. Cells were also subjected to DAPI staining for visualization of chromatin (blue). The immunofluoresence intensity of centrosomal NcoA4 and γ-tubulin at different mitotic stages was determined by image analysis and the ratio summarized in the bar graph for cells at different stages of cell division. Data are presented as mean ± SEM of 27–147 centrosomes per group. Bars with different letters are statistically different from one another as determined by Kruskal Wallis ANOVA for Ranks followed by Dunn's multiple comparison method (p
Figure Legend Snippet: Dynamic changes of NcoA4 association with the centrosome during mitotic progression. COS cells were stained for NcoA4 (red; B and E ), and γ-tubulin (green; A and D ) and examined under confocal microscopy. Merged images are shown in C and F and all images shown are of single optical slices. Cells were also subjected to DAPI staining for visualization of chromatin (blue). The immunofluoresence intensity of centrosomal NcoA4 and γ-tubulin at different mitotic stages was determined by image analysis and the ratio summarized in the bar graph for cells at different stages of cell division. Data are presented as mean ± SEM of 27–147 centrosomes per group. Bars with different letters are statistically different from one another as determined by Kruskal Wallis ANOVA for Ranks followed by Dunn's multiple comparison method (p

Techniques Used: Staining, Confocal Microscopy

NcoA4 association with the centrosome during mitotic progression. Representative images of COS cells at different stages of mitosis stained for NcoA4 (red; A, and D ), and γ−tubulin (green; B ), or Plk1 (green; E ). Merged images are shown in C and F . All images shown are of single optical slices obtained by confocal ( A to C ) or deconvolution ( D to F ) microscopy. Cells were also subjected to DAPI staining for visualization of chromatin (blue). NcoA4 was localized to aster formations (yellow arrows, A and C ) and centrosomes (white arrows, A and C ). Diminished NcoA4 staining at the centrosome during metaphase (red arrows, A and D ) using γ-tubulin ( C ) or Plk1 ( F ) as a centrosomal marker. [Bars: 20 µm].
Figure Legend Snippet: NcoA4 association with the centrosome during mitotic progression. Representative images of COS cells at different stages of mitosis stained for NcoA4 (red; A, and D ), and γ−tubulin (green; B ), or Plk1 (green; E ). Merged images are shown in C and F . All images shown are of single optical slices obtained by confocal ( A to C ) or deconvolution ( D to F ) microscopy. Cells were also subjected to DAPI staining for visualization of chromatin (blue). NcoA4 was localized to aster formations (yellow arrows, A and C ) and centrosomes (white arrows, A and C ). Diminished NcoA4 staining at the centrosome during metaphase (red arrows, A and D ) using γ-tubulin ( C ) or Plk1 ( F ) as a centrosomal marker. [Bars: 20 µm].

Techniques Used: Staining, Microscopy, Marker

21) Product Images from "1(OH) Vitamin D3 Supplementation Improves the Sensitivity of the Immune-Response during Peg-IFN/RBV Therapy in Chronic Hepatitis C Patients-Case Controlled Trial"

Article Title: 1(OH) Vitamin D3 Supplementation Improves the Sensitivity of the Immune-Response during Peg-IFN/RBV Therapy in Chronic Hepatitis C Patients-Case Controlled Trial

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063672

Comparison of Th1 and Tregs between 1(OH) vitamin D3/Peg-IFN/RBV and Peg-IFN/RBV. Representative dot plots of CD3 + CD4 + CD25 + IL7R − (Tregs) and CD3 + CD4 + CXCR3 + CCR5 + (Th1 cells) are shown. (A) Frequencies of Th1 and Tregs among the 4 groups (IL28B T/T vitamin D3/Peg-IFN/RBV, IL28B T/G or G/G vitamin D3/Peg-IFN/RBV, IL28B T/T Peg-IFN/RBV, and IL28B T/G or G/G Peg-IFN/RBV) are shown. (B) Comparison of the T-bet and IFN-γ mRNA expression between subjects treated with vitamin D3/Peg-IFN/RBV therapy and those treated with Peg-IFN/RBV therapy. Each group included 5 patients. Total mRNA was extracted from isolated CD4 + T cells. The relative expression levels are shown in bar graphs. The statistical analysis was carried out by independent student t -test.
Figure Legend Snippet: Comparison of Th1 and Tregs between 1(OH) vitamin D3/Peg-IFN/RBV and Peg-IFN/RBV. Representative dot plots of CD3 + CD4 + CD25 + IL7R − (Tregs) and CD3 + CD4 + CXCR3 + CCR5 + (Th1 cells) are shown. (A) Frequencies of Th1 and Tregs among the 4 groups (IL28B T/T vitamin D3/Peg-IFN/RBV, IL28B T/G or G/G vitamin D3/Peg-IFN/RBV, IL28B T/T Peg-IFN/RBV, and IL28B T/G or G/G Peg-IFN/RBV) are shown. (B) Comparison of the T-bet and IFN-γ mRNA expression between subjects treated with vitamin D3/Peg-IFN/RBV therapy and those treated with Peg-IFN/RBV therapy. Each group included 5 patients. Total mRNA was extracted from isolated CD4 + T cells. The relative expression levels are shown in bar graphs. The statistical analysis was carried out by independent student t -test.

Techniques Used: Expressing, Isolation

Cytokine profiles in the ex vivo and in vitro samples treated with vitamin D3. Sequential data of quantification of 3 cytokines (IFN-γ, IP-10 and RANTES) during 1(OH) vitamin D3 pre-treatment (pre vs 0w), 1(OH) vitamin D3/Peg-IFN/RBV therapy are shown (A). Dotted lines indicate the data of each subject. Black lines indicate the averaged data. Error bars indicate standard deviation. The data from IL28B (T/T) subjects or IL28B (T/G or G/G) subjects are shown in the independent graphs (A). Comparisons of the amounts of 3 cytokines (IFN-γ, IP-10 and RANTES) between the 1(OH) vitamin D3/PEG-IFN/RBV group (VitD3+standard of care (SOC)) and Peg-IFN/RBV group (SOC) at 0 weeks and 12 weeks after the start of Peg-IFN/RBV treatment are shown (B). Analysis of the changes in the amounts of the 3 cytokines (IFNγ, IP-10 and RANTES) during 12 weeks treatment of Peg-IFN/RBV is shown. Schema of in vitro -analysis of co-culture is shown (B). alfa-calcidol: 1(OH)vitamin D3 and calcitriol: 1,25(OH)vitamin D3 were used to analyze the cytokine production in vitro . Black bars indicate the data from samples treated with alfa-calcidol. Gray bars indicate the data from samples treated with calcitriol. *p
Figure Legend Snippet: Cytokine profiles in the ex vivo and in vitro samples treated with vitamin D3. Sequential data of quantification of 3 cytokines (IFN-γ, IP-10 and RANTES) during 1(OH) vitamin D3 pre-treatment (pre vs 0w), 1(OH) vitamin D3/Peg-IFN/RBV therapy are shown (A). Dotted lines indicate the data of each subject. Black lines indicate the averaged data. Error bars indicate standard deviation. The data from IL28B (T/T) subjects or IL28B (T/G or G/G) subjects are shown in the independent graphs (A). Comparisons of the amounts of 3 cytokines (IFN-γ, IP-10 and RANTES) between the 1(OH) vitamin D3/PEG-IFN/RBV group (VitD3+standard of care (SOC)) and Peg-IFN/RBV group (SOC) at 0 weeks and 12 weeks after the start of Peg-IFN/RBV treatment are shown (B). Analysis of the changes in the amounts of the 3 cytokines (IFNγ, IP-10 and RANTES) during 12 weeks treatment of Peg-IFN/RBV is shown. Schema of in vitro -analysis of co-culture is shown (B). alfa-calcidol: 1(OH)vitamin D3 and calcitriol: 1,25(OH)vitamin D3 were used to analyze the cytokine production in vitro . Black bars indicate the data from samples treated with alfa-calcidol. Gray bars indicate the data from samples treated with calcitriol. *p

Techniques Used: Ex Vivo, In Vitro, Standard Deviation, Co-Culture Assay

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Confocal Microscopy:

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Synthesized:

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Positron Emission Tomography-Computed Tomography:

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Construct:

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Electrophoresis:

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Incubation:

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Article Snippet: Reaction mixtures were then heated at 70°C for 15 min to inactivate RT and incubated with RNase H at 37°C for 20 min to remove RNA. .. IL-4, IFN-γ, and β-actin (housekeeping gene) cDNAs were amplified in a 480 Perkin-Elmer thermocycler with the following sequence-specific primers ( , ): IL-4, 5′-CTTCCCCCTCTGTTCTTCCT and 3′-TTCCTGTCGAGCCGTTTCAG; IFN-γ, 5′-AGTTATATCTTGGCTTTTCA and 3′-ACCGAATAATTAGTCAGCTT; β-actin, 5′-GTGGGGCGCCCCAGGCACCA and 3′-CTCCTTAATGTCACGCACGATTTC.

Article Title: Recombinant Complement Receptor 2 Radiolabeled with [99mTc(CO)3]+ : A Potential New Radiopharmaceutical for Imaging Activated Complement
Article Snippet: In the C3d+ red blood cell assay, 2.5×107 C3d+ RBCs and C3d- RBCs were incubated with 99m Tc-rCR2, K41E 99m Tc-CR2, 99m Tc-C2A or 10 µg/mL anti-human C3d followed by 99m Tc-rCR2. .. The percent of total radioactivity bound to C3d+ RBCs was determined as a ratio of radioactivity associated with the cell pellet, as determined by gamma counting (Wallac, 1282 COMPUGAMMA, PerkinElmer, UK), vs. total radioactivity added.

Radioactivity:

Article Title: Evaluation of an [18F]AlF-NOTA Analog of Exendin-4 for Imaging of GLP-1 Receptor in Insulinoma
Article Snippet: .. Radioactivity was determined using an on-line radioactivity detector or collection of 1-min fractions followed by gamma-counting (Wallach Wizard, PerkinElmer, Waltham, MA). .. Cell culture and animal model Studies with live animals were conducted in compliance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use of the Clinical Center, NIH.

Article Title: Intrinsic radiolabeling of Titanium-45 using mesoporous silica nanoparticles
Article Snippet: .. The radioactivity in the tissue was measured using a gamma-counter (Perkin-Elmer) and presented as%ID/g (%ID/g=percentage injected dose per gram, mean±SD). .. 45 Ti was produced by proton irradiation of a 0.25-mm-thick scandium foil (99.9%, 96–140 mg) with a current of 20 μA on a GE PETtrace.

Article Title: Functionalised Carbon Nanotubes Enhance Brain Delivery of Amyloid-Targeting Pittsburgh Compound B (PiB)-Derived Ligands
Article Snippet: .. The homogenates were centrifuged at 3220 x g (Eppendorf 5810R, Fisher Scientific UK) for 15 min to separate parenchyma (supernatant) and capillaries (pellet), and the radioactivity was measured using gamma counting (LKB Wallac 1282 Compugamma, PerkinElmer). .. Results (mean ± SD, n=3) are expressed as % of injected dose per brain fraction (% ID/brain fraction) followed by the analysis of brain parenchyma to blood ratio.

Article Title: Recombinant Complement Receptor 2 Radiolabeled with [99mTc(CO)3]+ : A Potential New Radiopharmaceutical for Imaging Activated Complement
Article Snippet: .. The percent of total radioactivity bound to C3d+ RBCs was determined as a ratio of radioactivity associated with the cell pellet, as determined by gamma counting (Wallac, 1282 COMPUGAMMA, PerkinElmer, UK), vs. total radioactivity added. .. Binding of 99m Tc-rCR2to C3d Binding of 99m Tc-rCR2 to C3d was evaluated by ELISA using a similar protocol as described above.

Activity Assay:

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain
Article Snippet: .. For each organ, the sample was weighed and activity was measured by gamma counting with a PerkinElmer Wizard 2480 (Waltham, MA, USA) to calculate %ID/g. .. For statistical analysis, a two-tailed Student’s t-test was used to compare the uptake of 52 Mn2+ at 4 and 48 hours and for the two contrast agent formulations.

Expressing:

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis
Article Snippet: .. Average intensity levels in GFP expressing cells were set as 1 and all other average intensity values were normalized accordingly. γ-tubulin and NEDD1 volumes were measured from Z stack images of cells based on either γ-tubulin or NEDD1 antibody staining using the volume measurements tool in Volocity6 image analysis package (PerkinElmer, Waltham, MA). ..

Ex Vivo:

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain
Article Snippet: Biodistribution of 52 Mn2+ was measured with ex vivo organ gamma counting in order to examine the uptake of both NCA and CA 52 Mn2+ in major organs of the rat. .. For each organ, the sample was weighed and activity was measured by gamma counting with a PerkinElmer Wizard 2480 (Waltham, MA, USA) to calculate %ID/g.

Article Title: Intrinsic radiolabeling of Titanium-45 using mesoporous silica nanoparticles
Article Snippet: Paragraph title: Ex vivo biodistribution study ... The radioactivity in the tissue was measured using a gamma-counter (Perkin-Elmer) and presented as%ID/g (%ID/g=percentage injected dose per gram, mean±SD).

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: .. Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA). .. Dynamic PET data were binned into 46 frames (12 × 5 s, 6 × 10 s, 6 × 30 s, 6 × 150 s, 6 × 300 s) and frames were reconstructed using non-scatter-corrected 3D ordered-subset expectation maximization followed by maximum a posteriori reconstruction (OSEM3D/MAP).

Kinase Assay:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: Paragraph title: Recombinant proteins and in vitro kinase assay ... One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ).

Transfection:

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis
Article Snippet: Transfected cells were identified using the 488 channel and z stack ranges were set using the centrosome markers. .. Average intensity levels in GFP expressing cells were set as 1 and all other average intensity values were normalized accordingly. γ-tubulin and NEDD1 volumes were measured from Z stack images of cells based on either γ-tubulin or NEDD1 antibody staining using the volume measurements tool in Volocity6 image analysis package (PerkinElmer, Waltham, MA).

Article Title: DAPK-ZIPK-L13a Axis Constitutes a Negative-Feedback Module Regulating Inflammatory Gene Expression
Article Snippet: .. 24 h after transfection with wild type or S77A mutant L13a-expressing plasmid DNA, cells were treated with IFN-γ for 20 h and then labeled with a 4-h pulse of 500 µCi [32 P]orthophosphate (Perkin-Elmer, Boston, MA) in phosphate free medium. .. Labeled cells were lysed in phosphosafe buffer and expressed Flag-L13a proteins were immunoprecipitated with anti-Flag antibody-conjugated agarose (Sigma) in detergent-containing RIPA buffer.

Immunoprecipitation:

Article Title: DAPK-ZIPK-L13a Axis Constitutes a Negative-Feedback Module Regulating Inflammatory Gene Expression
Article Snippet: 24 h after transfection with wild type or S77A mutant L13a-expressing plasmid DNA, cells were treated with IFN-γ for 20 h and then labeled with a 4-h pulse of 500 µCi [32 P]orthophosphate (Perkin-Elmer, Boston, MA) in phosphate free medium. .. Labeled cells were lysed in phosphosafe buffer and expressed Flag-L13a proteins were immunoprecipitated with anti-Flag antibody-conjugated agarose (Sigma) in detergent-containing RIPA buffer.

Generated:

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies
Article Snippet: The images shown in (panels A to C and G to I), 4, 5 (panels A to C and G to L), 6, 7, 8 (panels A to C) and 9 were generated by confocal microscopy using a Hamamatsu C9100-13 EM CCD camera with HCX PL APO (40X to 63X) objectives. .. The ratio of staining intensity for NcoA4 to that for γ-tubulin at the centrosome in cells at different mitotic phases was determined by Volocity Quantification software version 5.3 (Perkin Elmer, Woodbridge, ON, Canada).

Imaging:

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies
Article Snippet: Paragraph title: Immunofluorescence staining and imaging ... The ratio of staining intensity for NcoA4 to that for γ-tubulin at the centrosome in cells at different mitotic phases was determined by Volocity Quantification software version 5.3 (Perkin Elmer, Woodbridge, ON, Canada).

Article Title: Intrinsic radiolabeling of Titanium-45 using mesoporous silica nanoparticles
Article Snippet: Biodistribution studies were performed to confirm that the %ID/g values based on PET imaging truly represented the radioactivity distribution in tumor-bearing mice. .. The radioactivity in the tissue was measured using a gamma-counter (Perkin-Elmer) and presented as%ID/g (%ID/g=percentage injected dose per gram, mean±SD).

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: Paragraph title: Animal Model, PET/CT Imaging ... Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA).

Sequencing:

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis
Article Snippet: .. Total aortic RNA was isolated from 15-week-old E0 × CD4dnTβRII and E0 mice by using RNeasy (QIAGEN Inc., Valencia, California, USA), reverse-transcribed with Superscript-II (Life Technologies Inc., Rockville, Maryland, USA) and random hexamers, and amplified by real-time PCR using primers and probes for IFN-γ and hypoxanthine guanidine ribonucleosyltransferase (HPRT; sequences available on request) in an ABI 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, California, USA). .. Total RNA was analyzed by BioAnalyzer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) capillary electrophoresis.

Article Title: Increased Levels of Alternatively Spliced Interleukin 4 (IL-4?2) Transcripts in Peripheral Blood Mononuclear Cells from Patients with Systemic Sclerosis
Article Snippet: .. IL-4, IFN-γ, and β-actin (housekeeping gene) cDNAs were amplified in a 480 Perkin-Elmer thermocycler with the following sequence-specific primers ( , ): IL-4, 5′-CTTCCCCCTCTGTTCTTCCT and 3′-TTCCTGTCGAGCCGTTTCAG; IFN-γ, 5′-AGTTATATCTTGGCTTTTCA and 3′-ACCGAATAATTAGTCAGCTT; β-actin, 5′-GTGGGGCGCCCCAGGCACCA and 3′-CTCCTTAATGTCACGCACGATTTC. ..

Injection:

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain
Article Snippet: A target injection dose of 100 uCi was delivered to rats ( N = 3 per time point and per contrast agent formulation) via tail vein infusion at a rate of 2 mL/hr for an average of 8–10 minutes. .. For each organ, the sample was weighed and activity was measured by gamma counting with a PerkinElmer Wizard 2480 (Waltham, MA, USA) to calculate %ID/g.

Article Title: Intrinsic radiolabeling of Titanium-45 using mesoporous silica nanoparticles
Article Snippet: .. The radioactivity in the tissue was measured using a gamma-counter (Perkin-Elmer) and presented as%ID/g (%ID/g=percentage injected dose per gram, mean±SD). .. 45 Ti was produced by proton irradiation of a 0.25-mm-thick scandium foil (99.9%, 96–140 mg) with a current of 20 μA on a GE PETtrace.

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: Injected activities for dynamic PET scans (3.3 MBq) were increased relative to static PET scans to provide sufficient counting statistics during short-duration PET frames. .. Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA).

Article Title: Functionalised Carbon Nanotubes Enhance Brain Delivery of Amyloid-Targeting Pittsburgh Compound B (PiB)-Derived Ligands
Article Snippet: The homogenates were centrifuged at 3220 x g (Eppendorf 5810R, Fisher Scientific UK) for 15 min to separate parenchyma (supernatant) and capillaries (pellet), and the radioactivity was measured using gamma counting (LKB Wallac 1282 Compugamma, PerkinElmer). .. Results (mean ± SD, n=3) are expressed as % of injected dose per brain fraction (% ID/brain fraction) followed by the analysis of brain parenchyma to blood ratio.

Recombinant:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: Paragraph title: Recombinant proteins and in vitro kinase assay ... One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ).

Immunofluorescence:

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies
Article Snippet: Paragraph title: Immunofluorescence staining and imaging ... The ratio of staining intensity for NcoA4 to that for γ-tubulin at the centrosome in cells at different mitotic phases was determined by Volocity Quantification software version 5.3 (Perkin Elmer, Woodbridge, ON, Canada).

Article Title: Host Defense Mechanisms in Secondary Syphilitic Lesions
Article Snippet: Paragraph title: Immunofluorescence and Immunohistological Staining ... Cytokine stainings for IFN-γ and IL-17 were performed using a tyramide signal amplification (TSA) system (PerkinElmer, Waltham, MA) according to the manufacturer’s suggestions.

Animal Model:

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: Paragraph title: Animal Model, PET/CT Imaging ... Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA).

Fluorescence:

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis
Article Snippet: Average intensity levels in GFP expressing cells were set as 1 and all other average intensity values were normalized accordingly. γ-tubulin and NEDD1 volumes were measured from Z stack images of cells based on either γ-tubulin or NEDD1 antibody staining using the volume measurements tool in Volocity6 image analysis package (PerkinElmer, Waltham, MA). .. Centrosomes were located using PACT fluorescence and the total intensity of PCM1 or Cep290 was measured in an area of 0.85 µm2 around the centrosome.

Mutagenesis:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: Full-length and mutant Suv39h1 cDNA was subcloned into pMAL-HV vector. .. One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ).

Article Title: DAPK-ZIPK-L13a Axis Constitutes a Negative-Feedback Module Regulating Inflammatory Gene Expression
Article Snippet: .. 24 h after transfection with wild type or S77A mutant L13a-expressing plasmid DNA, cells were treated with IFN-γ for 20 h and then labeled with a 4-h pulse of 500 µCi [32 P]orthophosphate (Perkin-Elmer, Boston, MA) in phosphate free medium. .. Labeled cells were lysed in phosphosafe buffer and expressed Flag-L13a proteins were immunoprecipitated with anti-Flag antibody-conjugated agarose (Sigma) in detergent-containing RIPA buffer.

Isolation:

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis
Article Snippet: .. Total aortic RNA was isolated from 15-week-old E0 × CD4dnTβRII and E0 mice by using RNeasy (QIAGEN Inc., Valencia, California, USA), reverse-transcribed with Superscript-II (Life Technologies Inc., Rockville, Maryland, USA) and random hexamers, and amplified by real-time PCR using primers and probes for IFN-γ and hypoxanthine guanidine ribonucleosyltransferase (HPRT; sequences available on request) in an ABI 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, California, USA). .. Total RNA was analyzed by BioAnalyzer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) capillary electrophoresis.

Microscopy:

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies
Article Snippet: Images shown in (panels D to F and J to L), 5 (panels D to F) and 8 (panels D to F) were generated by deconvolution microscopy using a CoolSnap HQ CCD camera with Olympus lens N.A. .. The ratio of staining intensity for NcoA4 to that for γ-tubulin at the centrosome in cells at different mitotic phases was determined by Volocity Quantification software version 5.3 (Perkin Elmer, Woodbridge, ON, Canada).

Mouse Assay:

Article Title: PET/CT Based In Vivo Evaluation of 64Cu Labelled Nanodiscs in Tumor Bearing Mice
Article Snippet: .. Gamma counting Following the 48 hour PET scan, the mice were killed, blood collected and organs of interest harvested for endpoint measurements in a Wizard2 gamma-counter (Perkin Elmer, Skovlunde, Denmark) with a counting efficiency of 9.43% for 64 Cu. ..

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis
Article Snippet: .. Total aortic RNA was isolated from 15-week-old E0 × CD4dnTβRII and E0 mice by using RNeasy (QIAGEN Inc., Valencia, California, USA), reverse-transcribed with Superscript-II (Life Technologies Inc., Rockville, Maryland, USA) and random hexamers, and amplified by real-time PCR using primers and probes for IFN-γ and hypoxanthine guanidine ribonucleosyltransferase (HPRT; sequences available on request) in an ABI 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, California, USA). .. Total RNA was analyzed by BioAnalyzer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) capillary electrophoresis.

Article Title: Intrinsic radiolabeling of Titanium-45 using mesoporous silica nanoparticles
Article Snippet: Mice were euthanized, and blood, 4T1 tumor, and major organs/tissues were collected and wet-weighed. .. The radioactivity in the tissue was measured using a gamma-counter (Perkin-Elmer) and presented as%ID/g (%ID/g=percentage injected dose per gram, mean±SD).

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: Following imaging, mice were immediately sacrificed by CO2 asphyxiation, and organs were extracted. .. Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Increased Levels of Alternatively Spliced Interleukin 4 (IL-4?2) Transcripts in Peripheral Blood Mononuclear Cells from Patients with Systemic Sclerosis
Article Snippet: Paragraph title: RT-PCR. ... IL-4, IFN-γ, and β-actin (housekeeping gene) cDNAs were amplified in a 480 Perkin-Elmer thermocycler with the following sequence-specific primers ( , ): IL-4, 5′-CTTCCCCCTCTGTTCTTCCT and 3′-TTCCTGTCGAGCCGTTTCAG; IFN-γ, 5′-AGTTATATCTTGGCTTTTCA and 3′-ACCGAATAATTAGTCAGCTT; β-actin, 5′-GTGGGGCGCCCCAGGCACCA and 3′-CTCCTTAATGTCACGCACGATTTC.

Positron Emission Tomography:

Article Title: PET/CT Based In Vivo Evaluation of 64Cu Labelled Nanodiscs in Tumor Bearing Mice
Article Snippet: .. Gamma counting Following the 48 hour PET scan, the mice were killed, blood collected and organs of interest harvested for endpoint measurements in a Wizard2 gamma-counter (Perkin Elmer, Skovlunde, Denmark) with a counting efficiency of 9.43% for 64 Cu. ..

Article Title: Intrinsic radiolabeling of Titanium-45 using mesoporous silica nanoparticles
Article Snippet: Biodistribution studies were performed to confirm that the %ID/g values based on PET imaging truly represented the radioactivity distribution in tumor-bearing mice. .. The radioactivity in the tissue was measured using a gamma-counter (Perkin-Elmer) and presented as%ID/g (%ID/g=percentage injected dose per gram, mean±SD).

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: Injected activities for dynamic PET scans (3.3 MBq) were increased relative to static PET scans to provide sufficient counting statistics during short-duration PET frames. .. Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA).

Labeling:

Article Title: High-density lipoprotein endocytosis in endothelial cells
Article Snippet: Paragraph title: [3 H-CE-, 125 I-]-HDL labeling and uptake experiments ... [125 I-] radioactivity in the lysates was counted using a gamma-counter (COBRAII Auto-gamma; Perkin Elmer).

Article Title: Host Defense Mechanisms in Secondary Syphilitic Lesions
Article Snippet: Cytokine stainings for IFN-γ and IL-17 were performed using a tyramide signal amplification (TSA) system (PerkinElmer, Waltham, MA) according to the manufacturer’s suggestions. .. Labeled cells were enumerated per visual field and expressed as the number of epidermal cells per millimeter basement membrane as well as of cells per millimeter2 dermis ± SEM.

Article Title: DAPK-ZIPK-L13a Axis Constitutes a Negative-Feedback Module Regulating Inflammatory Gene Expression
Article Snippet: .. 24 h after transfection with wild type or S77A mutant L13a-expressing plasmid DNA, cells were treated with IFN-γ for 20 h and then labeled with a 4-h pulse of 500 µCi [32 P]orthophosphate (Perkin-Elmer, Boston, MA) in phosphate free medium. .. Labeled cells were lysed in phosphosafe buffer and expressed Flag-L13a proteins were immunoprecipitated with anti-Flag antibody-conjugated agarose (Sigma) in detergent-containing RIPA buffer.

Bradford Protein Assay:

Article Title: High-density lipoprotein endocytosis in endothelial cells
Article Snippet: [125 I-] radioactivity in the lysates was counted using a gamma-counter (COBRAII Auto-gamma; Perkin Elmer). .. Measurements were normalized to protein content, determined by the Bradford protein assay (Biorad, Germany).

Concentration Assay:

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain
Article Snippet: 52 Mn2+ was prepared either at a concentration of 0.33 mCi/mL (12.2 MBq/mL) in saline (NCA) or at an activity concentration of 0.37 mCi/mL (13.7 MBq/mL) in 66.7 mM MnCl2 . .. For each organ, the sample was weighed and activity was measured by gamma counting with a PerkinElmer Wizard 2480 (Waltham, MA, USA) to calculate %ID/g.

Article Title: High-density lipoprotein endocytosis in endothelial cells
Article Snippet: [3 H-CE-, 125 I]-HDL or [125 I]-HDL was added to each well at a final concentration of 10 μg/mL. .. [125 I-] radioactivity in the lysates was counted using a gamma-counter (COBRAII Auto-gamma; Perkin Elmer).

Purification:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: In vitro kinase reactions were performed by incubating CDK2/cyclin A2 or CDK2/cyclin E1 (Sigma-Aldrich) with purified bacterially expressed Suv39h1 proteins in a buffer containing 50 mM Tris-HCl, 10 mM MgCl2 , 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35 and 0.1 mM NaF for 1 h at 30°C. .. One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ).

Article Title: Host Defense Mechanisms in Secondary Syphilitic Lesions
Article Snippet: Cytokine stainings for IFN-γ and IL-17 were performed using a tyramide signal amplification (TSA) system (PerkinElmer, Waltham, MA) according to the manufacturer’s suggestions. .. Negative controls were obtained in all staining experiments by substituting isotype-matched IgG or IgM (purified, APC-, or FITC-labeled) for the primary antibody.

SDS Page:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ). .. The reactions were terminated with SDS sample buffer, boiled and resolved by SDS-PAGE gel.

Article Title: DAPK-ZIPK-L13a Axis Constitutes a Negative-Feedback Module Regulating Inflammatory Gene Expression
Article Snippet: 24 h after transfection with wild type or S77A mutant L13a-expressing plasmid DNA, cells were treated with IFN-γ for 20 h and then labeled with a 4-h pulse of 500 µCi [32 P]orthophosphate (Perkin-Elmer, Boston, MA) in phosphate free medium. .. Proteins in the immunoprecipitates were resolved by 12% SDS-PAGE and bands were visualized by autoradiography.

Plasmid Preparation:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: Full-length and mutant Suv39h1 cDNA was subcloned into pMAL-HV vector. .. One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ).

Article Title: DAPK-ZIPK-L13a Axis Constitutes a Negative-Feedback Module Regulating Inflammatory Gene Expression
Article Snippet: .. 24 h after transfection with wild type or S77A mutant L13a-expressing plasmid DNA, cells were treated with IFN-γ for 20 h and then labeled with a 4-h pulse of 500 µCi [32 P]orthophosphate (Perkin-Elmer, Boston, MA) in phosphate free medium. .. Labeled cells were lysed in phosphosafe buffer and expressed Flag-L13a proteins were immunoprecipitated with anti-Flag antibody-conjugated agarose (Sigma) in detergent-containing RIPA buffer.

Software:

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies
Article Snippet: .. The ratio of staining intensity for NcoA4 to that for γ-tubulin at the centrosome in cells at different mitotic phases was determined by Volocity Quantification software version 5.3 (Perkin Elmer, Woodbridge, ON, Canada). .. For this study, the area occupied by the centrosomes was defined by γ-tubulin staining which was used as template to measure the corresponding NcoA4 staining intensity.

Real-time Polymerase Chain Reaction:

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis
Article Snippet: .. Total aortic RNA was isolated from 15-week-old E0 × CD4dnTβRII and E0 mice by using RNeasy (QIAGEN Inc., Valencia, California, USA), reverse-transcribed with Superscript-II (Life Technologies Inc., Rockville, Maryland, USA) and random hexamers, and amplified by real-time PCR using primers and probes for IFN-γ and hypoxanthine guanidine ribonucleosyltransferase (HPRT; sequences available on request) in an ABI 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, California, USA). .. Total RNA was analyzed by BioAnalyzer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) capillary electrophoresis.

Binding Assay:

Article Title: High-density lipoprotein endocytosis in endothelial cells
Article Snippet: To calculate unspecific binding, a 40-fold excess of unlabeled HDL was added to every fourth well. .. [125 I-] radioactivity in the lysates was counted using a gamma-counter (COBRAII Auto-gamma; Perkin Elmer).

Article Title: Recombinant Complement Receptor 2 Radiolabeled with [99mTc(CO)3]+ : A Potential New Radiopharmaceutical for Imaging Activated Complement
Article Snippet: Paragraph title: Binding of 99m Tc-rCR2 to C3d+ cells ... The percent of total radioactivity bound to C3d+ RBCs was determined as a ratio of radioactivity associated with the cell pellet, as determined by gamma counting (Wallac, 1282 COMPUGAMMA, PerkinElmer, UK), vs. total radioactivity added.

Multicolor Immunofluorescence Staining:

Article Title: Host Defense Mechanisms in Secondary Syphilitic Lesions
Article Snippet: Single and multicolor immunofluorescence staining procedures were performed on frozen sections of patients with secondary syphilis ( n = 14) and healthy controls ( n = 10) as previously described. .. Cytokine stainings for IFN-γ and IL-17 were performed using a tyramide signal amplification (TSA) system (PerkinElmer, Waltham, MA) according to the manufacturer’s suggestions.

In Vitro:

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression
Article Snippet: Paragraph title: Recombinant proteins and in vitro kinase assay ... One microliter of 10 μCi gamma-ATP (Perkin-Elmer Corp., Norwalk, CT) was used ( , ).

Homogenization:

Article Title: Functionalised Carbon Nanotubes Enhance Brain Delivery of Amyloid-Targeting Pittsburgh Compound B (PiB)-Derived Ligands
Article Snippet: Briefly, each brain was homogenised in a glass homogeniser containing 1 mL of ice-cold depletion buffer (10 mM HEPES in HBSS, pH 7.4) via 15 stokes of the pestle, followed by addition of 1.6 mL of depletion buffer containing 26% dextran (148 kDa) and repeated pestle homogenisation (3 strokes). .. The homogenates were centrifuged at 3220 x g (Eppendorf 5810R, Fisher Scientific UK) for 15 min to separate parenchyma (supernatant) and capillaries (pellet), and the radioactivity was measured using gamma counting (LKB Wallac 1282 Compugamma, PerkinElmer).

Two Tailed Test:

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain
Article Snippet: For each organ, the sample was weighed and activity was measured by gamma counting with a PerkinElmer Wizard 2480 (Waltham, MA, USA) to calculate %ID/g. .. For statistical analysis, a two-tailed Student’s t-test was used to compare the uptake of 52 Mn2+ at 4 and 48 hours and for the two contrast agent formulations.

Activation Assay:

Article Title: Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport
Article Snippet: Due to the impact of volatile anesthetics on voltage-dependent calcium channel (VDCC) activation , , the second group (n = 3) received an intravenous (I.V.) bolus of 51 Mn2+ (1.6 MBq, 200 µl, 10% 0.01 M NaOAc/90% PBS) while awake. .. Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, MA).

Quantitation Assay:

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis
Article Snippet: Paragraph title: Quantitation of mRNA. ... Total aortic RNA was isolated from 15-week-old E0 × CD4dnTβRII and E0 mice by using RNeasy (QIAGEN Inc., Valencia, California, USA), reverse-transcribed with Superscript-II (Life Technologies Inc., Rockville, Maryland, USA) and random hexamers, and amplified by real-time PCR using primers and probes for IFN-γ and hypoxanthine guanidine ribonucleosyltransferase (HPRT; sequences available on request) in an ABI 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, California, USA).

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis
Article Snippet: Paragraph title: Data quantitation ... Average intensity levels in GFP expressing cells were set as 1 and all other average intensity values were normalized accordingly. γ-tubulin and NEDD1 volumes were measured from Z stack images of cells based on either γ-tubulin or NEDD1 antibody staining using the volume measurements tool in Volocity6 image analysis package (PerkinElmer, Waltham, MA).

Staining:

Article Title: Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies
Article Snippet: .. The ratio of staining intensity for NcoA4 to that for γ-tubulin at the centrosome in cells at different mitotic phases was determined by Volocity Quantification software version 5.3 (Perkin Elmer, Woodbridge, ON, Canada). .. For this study, the area occupied by the centrosomes was defined by γ-tubulin staining which was used as template to measure the corresponding NcoA4 staining intensity.

Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis
Article Snippet: .. Average intensity levels in GFP expressing cells were set as 1 and all other average intensity values were normalized accordingly. γ-tubulin and NEDD1 volumes were measured from Z stack images of cells based on either γ-tubulin or NEDD1 antibody staining using the volume measurements tool in Volocity6 image analysis package (PerkinElmer, Waltham, MA). ..

Article Title: Host Defense Mechanisms in Secondary Syphilitic Lesions
Article Snippet: Paragraph title: Immunofluorescence and Immunohistological Staining ... Cytokine stainings for IFN-γ and IL-17 were performed using a tyramide signal amplification (TSA) system (PerkinElmer, Waltham, MA) according to the manufacturer’s suggestions.

T-Test:

Article Title: Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain
Article Snippet: For each organ, the sample was weighed and activity was measured by gamma counting with a PerkinElmer Wizard 2480 (Waltham, MA, USA) to calculate %ID/g. .. For statistical analysis, a two-tailed Student’s t-test was used to compare the uptake of 52 Mn2+ at 4 and 48 hours and for the two contrast agent formulations.

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  • 99
    PerkinElmer γ 32 p atp
    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 <t>P]-ATP.</t> Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/PerkinElmer
    Average 99 stars, based on 10 article reviews
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    99
    PerkinElmer gamma counter
    Blood clearance of the 99m Tc-RGD4CβL. Three Wistar rats were injected with the 99m Tc-RGD4CβL. Blood was drawn at different time-points, and radioactivities were measured by a <t>gamma</t> counter, data are shown as %ID/g, T 1/2 α and T 1/2 β were 7.8 and 21.9 min respectively.
    Gamma Counter, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gamma counter/product/PerkinElmer
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    gamma counter - by Bioz Stars, 2020-02
    99/100 stars
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    Image Search Results


    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Journal: Biochemical pharmacology

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    doi: 10.1016/j.bcp.2013.10.015

    Figure Lengend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Article Snippet: Different amount of His-PXR as indicated (1 µg or 2.5 µg) was incubated in kinase buffer with 20 ng Cdk2/cyclin E (EMD Millipore, Billerica, MA), 5 µCi [γ-32 P]ATP (Perkin-Elmer, Santa Clara, CA), and 5 µM cold ATP.

    Techniques: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: Radiolabeled RNAs were prepared with γ-32 P-ATP (PerkinElmer) and T4 PNK (New England Biolabs) and purified by 20% denaturing PAGE followed by ethanol precipitation.

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Journal: Molecular Biology of the Cell

    Article Title: ?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    doi: 10.1091/mbc.E10-07-0636

    Figure Lengend Snippet: Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Article Snippet: In Vitro Kinase Assays pET and pET-Aly2 were incubated for 30 min at 30°C in kinase buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 , 1 mM DTT, 1 μM unlabeled ATP, aprotinin, and leupeptin) with 75 nM [γ-32 P]ATP (Perkin Elmer-Cetus) with or without Npr1 kinase (purified from Y258 yeast cells).

    Techniques: Expressing, Copurification, Western Blot, Fluorescence, Microscopy, Purification, Positron Emission Tomography, Incubation, SDS Page, Staining

    Blood clearance of the 99m Tc-RGD4CβL. Three Wistar rats were injected with the 99m Tc-RGD4CβL. Blood was drawn at different time-points, and radioactivities were measured by a gamma counter, data are shown as %ID/g, T 1/2 α and T 1/2 β were 7.8 and 21.9 min respectively.

    Journal: International Journal of Molecular Sciences

    Article Title: A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy

    doi: 10.3390/ijms16059625

    Figure Lengend Snippet: Blood clearance of the 99m Tc-RGD4CβL. Three Wistar rats were injected with the 99m Tc-RGD4CβL. Blood was drawn at different time-points, and radioactivities were measured by a gamma counter, data are shown as %ID/g, T 1/2 α and T 1/2 β were 7.8 and 21.9 min respectively.

    Article Snippet: Portions of 1 mL sodium hydroxide (1 mol/L) were added and the plates were incubated at room temperature for 1 h. The lysates were then collected and radioactivities were measured by a gamma counter (2470 WIZARD2, PerkinElmer, Waltham, MA, USA).

    Techniques: Injection