By P. Michael Conn
This new quantity of Methods in Enzymology keeps the legacy of this optimal serial with caliber chapters authored by way of leaders within the box. this is often the second of 2 volumes on endosome signaling and comprises chapters on such themes as dimension of access into the endosomal compartment by means of multi-parametric photograph research, evaluation of peptide internalization and endosomal signaling, and VEGF-A in endosomal signaling.
- Continues the legacy of this ultimate serial with caliber chapters authored by means of leaders within the box
- Covers endosome signaling
- Contains chapters on such themes as size of organic results of endosomal proteolysis of internalized insulin and multi-vesicular endosome biogenesis.
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Extra info for Endosome Signaling Part B
Incubate these cells at 37 C with CO2 for overnight. 4. Replace the transfection media with phenol red-free DMEM supplemented with 2% FBS. 5. 4 oil objective lens. The reconstituted YFP signals (Fig. , 2012). Study Ras in Endosomes by BiFC 33 3. 1. Create “baits” and negative controls We used Yn-H-Ras(G12V) as the bait. In addition to Yn-H-Ras△E (G12V), we also used Yn-GST as a negative control, in case some effectors still retain a weak binding to H-Ras△E(G12V). To reduce background and improve reproducibility, we found it helpful when the bait is stably expressed as follows: 1.
J. , & Posner, B. I. (2000). Association of phosphatidylinositol with the insulin receptor: Compartmentation in rat liver. American Journal of Physiology Endocrinology and Metabolism, 279, E266–E274. , & Authier, F. (2010). Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces ADP-ribosylating and apoptotic fragments. The FEBS Journal, 277, 3735–3749. , & Authier, F. (2007). Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity.
When the proteins of interest form a complex, the fragments of the fluorescent protein also refold to restore fluorescence, which can be readily detected by both FACS (Fig. 1B) and fluorescence microscopy (Fig. 1C). 1 Detecting H-Ras and Raf-RBD interaction by BiFC. (A) A schematic representation of the BiFC system—see text for details. (B) Yc-tagged Raf-RBD was cotransfected with indicated Yn-tagged constructs into HT1080 cells. The transfected cells were cultured for another 36 h before being examined by FACS analysis for YFP.