By P. Michael Conn
This new quantity of Methods in Enzymology maintains the legacy of this most excellent serial with caliber chapters authored through leaders within the box. this is often the second of 2 volumes on endosome signaling and comprises chapters on such subject matters as dimension of access into the endosomal compartment by way of multi-parametric snapshot research, overview of peptide internalization and endosomal signaling, and VEGF-A in endosomal signaling.
- Continues the legacy of this most suitable serial with caliber chapters authored through 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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Additional info for Endosome Signaling Part B
If using four plates/condition, add 2 mL of lysis buffer to the first plate, scrape cells, and transfer to the second plate. Scrape the second plate and transfer to the third plate. Continue this process until all four plates per given condition have been lysed. 5 mL) to a 15 mL conical tube. The cell lysate should be very viscous. 6. Repeat the previously mentioned lysis procedure for each of the other conditions and place into separate 15 mL conical tubes. 7. Homogenize the cell lysate with three 10-s bursts in a Polytron tissue grinder (settings 7–8).
Incubate at 4 C for 2 h on a gently rocking platform. 7. 5% BSA/KRH three times. 5% BSA/KRH and add to the cells. All subsequent steps must now be completed in the DARK. Incubate the cells at 4 C for 1 h on a gently rocking platform. 8. 5% BSA/KRH. , 30 min and 60 min). The incubation at 37 C permits the cell-surfacelabeled receptors to internalize. 9. The fixation procedure is as follows: i. Gently rinse coverslips five times with PBS. ii. Fix cells by adding 4% paraformaldehyde/PBS and incubate for 10 min at room temperature.
Et al. (2004). 1: A platform for reverse proteomics. Genome Research, 14, 2128–2135. , & Miyawaki, A. (2000). Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Research, 28, E78. , & Di Fiore, P. P. (2012). Endocytosis and signaling: Cell logistics shape the eukaryotic cell plan. Physiological Reviews, 92, 273–366. Tall, G. , Barbieri, M. , Stahl, P. , & Horazdovsky, B. F. (2001). Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1.
Endosome Signaling Part B by P. Michael Conn