Technical Reference #1836

1836.

Ubiquitin C-terminal Hydrolase-L1 Protects Cystic Fibrosis Transmembrane Conductance Regulator from Early Stages of Proteasomal Degradation Mark J. Henderson; Neera Vij; and Pamela L. Zeitlin, Johns Hopkins School of Medicine, Journal of Biological Chemistry, 285(1836), (2010)
Link To Paper

Keywords:
Diseases/Cystic Fibrosis; Proteases/Ubiquitination; Protein/Intracellular Trafficking; Protein/Targeting; RNA/RNAi

Materials & Methods:
Confocal Microscopy—IB3-1 cells were plated on 10-cm2 glass bottom dishes (Mattek Ashland MA) at 20% (immunolocalization) or 40% (fluorescent tag) confluence and allowed to grow overnight. For the mCherry-UCH-L1 and EmGFP experiments IB3-1 cells were transiently transfected for 24 h. The cells were fixed at room temperature with 4% paraformaldehyde or at 220 °C with ice-cold methanol for 30 min. For immunolocalization experiments the cells were fixed in fresh 4% paraformaldehyde for 25 min. The cells were then permeabilized in 0.2% Triton X-100 for 15 min blocked in 2% bovine serum albumin with PBS for 40 min and incubated with primary antibody overnight at 4°. The primary antibodies used were polyclonal UCH-L1 at 1:50 (Millipore) monoclonal RPS6 at 1:50 (Cell Signaling Danvers MA) and monoclonal KDEL at 1:50 (Abcam). Highly cross-adsorbed Alexa Fluor 488 and 568 (Invitrogen) secondary antibodies were used at 1:400 in 2% bovine serum albumin with PBS 1 10% goat serum.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
IB3-1 cells