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发布于:2020-1-9 18:37:10  访问:24 次 回复:0 篇
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Rac/Cdc42, throughout phagocytosis of eight.3 mm IgG beads. Just before phagocytosis, cells
130964-39-5 Formula internalized beads are indicated with a star. In sharp contrast, cells taken care of with LY294002 displayedyellow fluorescent protein (YFP), a fluorescent biosensor of Rac/ Cdc42 action consisting on the p21-binding domain (PBD) of p21-activated 67392-87-4 Purity & Documentation kinase (PAK)nine,16. Phagocytosis was then allowed to move forward for one, 3 or 8 min in advance of fixation and marking of F-actin with phalloidin-568 (Fig. As anticipated, internet sites of particle PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/23907221 engagement displayed prominent PAK(PBD)?YFP enrichment (akin to sites of GTP-bound Rac and/142880-36-2 MedChemExpress aControl (DMSO) one min+ eight.3-m IgG-opsonized beads 3 min eight minc30 s Control+ 1.6-m IgG-opsonized beads 2 min five min******b1 min + LY294002 3 min eight mind30 s + LY294002 2 min five min***ePAK(PBD) FPIgG beads (eight.3 )DMSOPhalloidin-Phalloidin-1 min3 minPAK(PBD) FP PAK(PBD) FPPhalloidin-PAK(PBD) FPPAK(PBD)-YFP8 minf+ LYPAK(PBD) FPPhalloidin-1 minPhalloidin-3 minPhalloidin-8 mingRac1(Q61L) RtailjLifeact + beads (all) Lifeact + beads (out)* **4.0 Whole number of beads involved for every cell (common) three.5 three.0 2.5 2.0 one.five one.0 0.five 0 n.s.* = P 0.05 ** = P 0.005 n.s.** *80 60 40 20C dc tro 4 one( two(G l Q sixty one 12V L) ).Rac/Cdc42, for the duration of phagocytosis of 8.three mm IgG beads. Right before phagocytosis, cells were being addressed with DMSO (e) or LY294002 (f) for ten min. Phagocytosis was permitted to progress with the indicated occasions, in advance of correcting and staining F-actin with phalloidin. Insets (boxed locations) clearly show magnified sights from the phagocytic cup. (g ) Confocal micrographs of Uncooked 264.7 macrophages transiently co-expressing Lifeact RFP in combination with constitutively lively Rac1 (g), constitutively lively Cdc42 (h) or perhaps a recruitable method of the Rac1 GEF Tiam1 (i). Addition of rapamycin triggers translocation of Tiam1 to your plasmalemma, where its rapamycin-binding domain (FKBP) interacts which has a next, complementary rapamycin-binding moiety. Transfectants ended up challenged with eight.3-mm IgG beads, and phagocytosis allowed to move forward for ten min before fixation. All phagocytic targets have been stained with Cy5-conjugated secondary antibody (shown in blue) ahead of phagocytosis. Extracellular beads were being identified by staining preset (non-permeabilized) cells having an Alexa Fluor 488-conjugated secondary antibody (revealed in green). Internalized beads are indicated with a star. Scale bar, ten mm. (j) Quantification of phagocytic indices (complete variety of beads linked for each mobile; black bars) and phagocytic efficiencies (ratio of internalized-to-total number of beads per cell; white bars) to the experiments explained in (g ). Values represent the suggests of three impartial replicates .e.m. A minimum of 25 cells ended up assessed for every replicate. *Pr0.05, **Pr0.005 or n.s. (not major) utilizing Student‘s two-tailed unpaired t-tests.Nature COMMUNICATIONS | 6:8623 | DOI: 10.1038/ncomms9623 | www.nature.com/naturecommunications2015 Macmillan Publishers Confined. All legal rights reserved.Mother nature COMMUNICATIONS | DOI: ten.1038/ncommsARTICLEor Cdc42) and colocalized with sites of powerful F-actin staining (1 min time stage in Fig. 1e; strong arrow).
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