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  1. Detailansicht für Treffer 1 öffnen
    Figure 2—figure supplement 1. Primary data from siRNA Screen 1 Human A549 cells were transfected with the indicated siRNA pools (Dharmacon) for 72 hr. ; Cells were lysed and immunoblotted for the indicated antibodies and immunoblots developed using the LI-COR Odyssey CLx Western Blot imaging system. The numbering system corresponds to that employed in Supplementary file 1. The intensities were quantified and are presented as pRab10/Total Rab10 ratio relative to scrambled siRNA control in Supplementary file 1.
    2019
    unknown
    Wie komme ich dran?
  3. Detailansicht für Treffer 3 öffnen
    Figure 2. siRNA screens to identify phosphatases that regulate Rab10 phosphorylation. ; (A) Workflow of siRNA knockdown screens employed in this study. Human A549 cells were transfected with siRNA pools (Dharmacon) for 72 hr targeting 264 phosphatase catalytic subunits (189 protein phosphatases, 26 halo acid dehalogenase phosphatases, 18 chloroperoxidases, five nucleoside-diphosphate-linked moiety-X phosphatases and seven carbohydrate phosphatases) as well as 56 characterized regulatory subunits. The list of phosphatase components targeted, and oligonucleotide sequences utilized is provided in Supplementary file 1. Cells were lysed and immunoblotted for total LRRK2, LRRK2 pS935, total Rab10, and Rab10 pT73 and immunoblots developed using the LI-COR Odyssey CLx Western Blot imaging system. The ratio of phospho-Thr73 Rab10/total Rab10 in each sample was quantified using the Image Studio software. (B) Summary of results from Screen 1 and 2 (without MLi-2 pretreatment). Calculated ratio of phosphorylated Rab10 and total Rab10 relative to the scrambled siRNA control, ranked from highest increase in Rab10 phosphorylation to the strongest decrease (mean of the two replicates, SD values not shown on the chart, numerical data provided in Supplementary file 1). (C) As in (B), summary of results from Screen 3 (in which cells were treated for 5 min with 100 nM MLi-2 treatment prior to cell lysis), ratio calculated relative to scrambled siRNA control without MLi-2 treatment. (D) Human A549 cells were transfected with siRNA for 72 hr with the indicated top hits from screen 1 and 2 that increased Rab10 phosphorylation. Cells were then lysed and immunoblotted with the indicated antibodies, analyzed as described above. Data are presented relative to the ratio of Rab10 phosphorylation/total Rab10 observed in cells treated with scrambled siRNA as mean ± SD. (E) As in (D) except the key hits that increased Rab10 phosphorylation in Screen three were reanalyzed. Cells were also treated with 100 nM MLi-2 prior to cell lysis. (F) As in (D) except the key hits that decreased Rab10 phosphorylation in Screen 1 to 3 were reanalyzed.
    2019
    unknown
    Wie komme ich dran?
  4. Detailansicht für Treffer 4 öffnen
    Figure 2—figure supplement 2. Primary data from siRNA Screen 2 Human A549 cells were transfected with the indicated siRNA pools (Dharmacon) for 72 hr. ; Cells were lysed and immunoblotted for the indicated antibodies and immunoblots developed using the LI-COR Odyssey CLx Western Blot imaging system. The numbering system corresponds to that employed in Supplementary file 1. The intensities were quantified and are presented as pRab10/Total Rab 10 ratio relative to scrambled siRNA control in Supplementary file 1.
    2019
    unknown
    Wie komme ich dran?
  5. Detailansicht für Treffer 5 öffnen
    Figure 9—figure supplement 1. PPM1H[D288A] substrate trapping mutant stably interacts with LRRK2 phosphorylated Rab10 protecting it from dephosphorylation. ; (A) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G] and either wild-type HA-PPM1H or substrate trapping HA-PPM1H[D288A] mutant. 24 hr post-transfection, cells were treated with ±200 nM MLi-2 for 90 min lysed and subjected to a HA-immunoprecipitation and analyzed by Phos-tag immunoblotting with the indicated antibodies (1 µg/ml). Each lane represents cell extract obtained from a different dish of cells (three replicates per condition without MLi-2 treatment, one replicate per condition with MLi-2 treatment). Membranes were developed using ECL. (B) HEK293 cells were transiently transfected with constructs expressing PPM1H[D288A] with either wild-type or indicated pathogenic or catalytically inactive LRRK2. 24 hr post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies (1 µg/ml). Membranes were developed using Odyssey CLx Western Blot imaging. Each lane represents biological replicates (three replicates per condition). (C) As in (B) except that HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G] and either wild-type HA-PPM1H or indicated HA-PPM1H mutants or empty HA-vector for no phosphatase control. 24 hr post-transfection cells were treated ±200 nM MLi-2 prior to cell lysis for 90 min.
    2019
    unknown
    Wie komme ich dran?
  6. Detailansicht für Treffer 6 öffnen
    Figure 9. Identification of a substrate trapping mutant of PPM1H that forms a stable interaction with endogenous LRRK2 phosphorylated Rab8A and Rab10. ; (A) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G] and either wild-type HA-PPM1H or the PPM1H[D288A] mutant, as well as the corresponding mutants of the closely related PPM1J[D279A] and PPM1M[D235A]. 24 hr post-transfection cells were lysed and subjected to a HA-immunoprecipitation and analyzed by immunoblotting with the indicated antibodies (1 µg/ml). Membranes were developed using Odyssey CLx Western Blot imaging. Each lane represents cell extract obtained from a different dish of cells (three replicates per condition). (B) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G] and either wild-type HA-PPM1H or the HA-PPM1H[D288A] mutant, lysed and subjected to a HA-immunoprecipitation and total immunoprecipitates were subjected to ‘on-bead’ digestion using trypsin following multiplexed TMT labeling the samples were pooled and fractionated into four fractions and analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer in MS3 mode. The raw data was processed using MaxQuant pipeline and protein groups were further processed using Perseus software suite. The x-axis of the volcano plot represents the differential enrichment between HA-PPM1H[D288A]+LRRK2[R1441G] and wild-type HA-PPM1H+LRRK2[R1441G] and the y-axis represents the permutation-based false discovery rate corrected significance of a two tailed t-test. The differential enriched protein groups are highlighted in red filled circles along with the protein groups that are not changing are highlighted in blue with their gene names. The list of protein groups is included in Supplementary file 3.
    2019
    unknown
    Wie komme ich dran?
  7. Detailansicht für Treffer 7 öffnen
    Figure 2—figure supplement 3. Primary data from siRNA Screen 3 Human A549 cells were transfected with the indicated siRNA pools (Dharmacon) for 72 hr. ; Cells were treated for 5 min with 100 nM MLi-2, lysed and immunoblotted for the indicated antibodies and immunoblots developed using the LI-COR Odyssey CLx Western Blot imaging system. The numbering system corresponds to that employed in Supplementary file 1. The intensities were quantified and are presented as pRab10/Total Rab10 ratio relative to scrambled siRNA control without MLi-2 treatment in Supplementary file 1.
    2019
    unknown
    Wie komme ich dran?
  9. Detailansicht für Treffer 9 öffnen
    Figure 5—figure supplement 1. Deglycosylated gp120 loses its binding specificity to SIGN-R1+ macrophages. ; Confocal microscopy of thick LN sections overlaid with fluorescent gp120, which is treated with PNGase F or not, and immunostained for the indicated markers. (A) LN section image (sagittal, tiled) shows control gp120, green; CD169, cyan; CD21/35, gray; F4/80, blue and SIGN-R1, red. Scale bar is 200 μm (top). (B) LN section image (sagittal, tiled) shows deglycosylated gp120, green; CD169, cyan; CD21/35, gray; F4/80, blue and SIGN-R1, red. Scale bar is 200 μm (top). Zoomed images of the white boxed area in (A) and (B) were shown in middle and bottom panels. Image in middle left shows gp120, green; CD169, cyan; CD21/35, gray; F4/80, blue and SIGN-R1, red. Image in middle right shows gp120, green and CD169, cyan. Image in bottom left shows CD169 and SIGN-R1, red. Image in bottom right shows the generated channel (yellow) of colocalization between gp120 and SIGN-R1. Scale bars are 100 μm. (C) Percentage of ROI colocalized area in bottom left panel in (A) and (B) were compared in graph. (D) The strength of fluorochrome after PNGase F treatment was measured by Odyssey CLx Infrared Imaging System. The numbers on top of each lanes indicated the amount of loaded gp120. The numbers on bottom of each lanes indicated the relative score of strength of fluorochrome on gp120.
    2015
    unknown
    Wie komme ich dran?
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