Ptdins4p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization"
Article Title: Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization
Journal: The Journal of Immunology Author Choice
Figure Legend Snippet: ARF6 is copolarized with PM PtdIns4P in neutrophils attaching to activated endothelial cells. ( A . The grid scales are 1 μm. ( B ) ARF6 is not polarized and minimally colocalized with PM PtdIns4P in mouse neutrophils attached to PK-coated surface under shear flow. Mouse neutrophils were transfected with GFP-P4M and ARF6-TagRFP plasmids and cultured for 6 h, then flowed through a chamber coated with polylysine at the rate of 1 dyn/cm 2 . The grid scales are 1 μm. ( C ) Quantification of colocalization of P4M and ARF6 in (A) and (B) conditions is shown as the Pearson coefficient. Each data point represents a cell. Data are presented as means ± SEM (Student t test). The experiments were repeated three times. **** p
Techniques Used: Transfection, Cell Culture
Figure Legend Snippet: ARF6 is a coincidence-detection code for RPH3A protein. ( A ) GTP-bound ARF6 augments the interaction of RPH3A with PtdIns4P. Purified recombinant myristoylated ARF6 protein preloaded with GDPγS or GTPγS was incorporated into phosphocholine (PC)/phosphatidylserine (PS) liposome containing PtdIns4P. The liposomes were then incubated with purified recombinant RPH3A protein, followed by density ultracentrifugation. Input and liposome-associated proteins were detected by Western analysis. ( B ) RPH3A preferentially binds to GTP-bound ARF6. Purified recombinant myristoylated ARF6 protein preloaded with GTPγS or GDPγS was incubated with purified His-tagged recombinant RPH3A protein, followed by nickel-bead pull-down and Western analysis. ( C ) ARF6 preferentially binds to p-RPH3A. Purified recombinant proteins were used in an His–pull-down assay under the conditions described in the figure. Proteins were then detected by Western blot analysis. ( D ) ARF6 augments the interaction between the RPH3A and RAB21. Purified GTPγS-bound myristoylated ARF6 and RAB21 were incubated with p-RPH3A and subjected to an His–pull-down assay. The proteins were detected by Western blot analysis.
Techniques Used: Purification, Recombinant, Incubation, Western Blot, Pull Down Assay