Biomedical Engineering Reference
In-Depth Information
could still be checked to ensure that tagging was not grossly
disruptive. One of these is cellular localization, which is quite
specifi c for some, but not all Rabs, and could be readily verifi ed
by confocal imaging (
see
Subheading
3.2
below). If the Rab
has known interacting partners and effectors, biochemical
approaches (
see
Subheading
3.3
below) could also be used to
verify integrity of function upon tagging.
1. Seed cells on glass coverslips such that it is ~30-40 % confl uent
the next day.
2. Transfect cells with siRNA using a suitable transfection reagent
(such as Lipofectamine
®
RNAiMAX Reagent (Life Technologies)
or infection with viral particles generated with retro- or lentivi-
ral constructs). The degree of silencing can be estimated by
Western immunoblot as described in Subheading
3.3
below or
by quantitative reverse transcription polymerase chain reaction
(RT-PCR) (
see
Note 4
).
3.1.2 Manipulation
of Rab Levels with RNAi
Silencing/Rescue
Experiments
3. Perform imaging analysis as outlined in Subheading
3.2
below.
4.
Rescue experiments
: the nature of the rescue construct would
vary depending on the nature of the silencing method (
see
Note 5
). Typically, rescues are done by transfecting or infect-
ing siRNA-/shRNA-treated cells for 24 h with expression
constructs of a non-RNAi degradable form of the Rab (or
infected with viruses expressing such) and the reversal of
phenotype assessed as in Subheading
3.2
below.
1. Seed cells on glass coverslips and grow to ~60-80 % confl u-
ency. Serum-starve cells overnight in basal DMEM.
2. Incubate cells with 0.25-0.5
3.2 Confocal
Imaging Analysis of
Ligand-Bound EGFR
g/ml of Texas Red (TxR) or
FITC-tagged epidermal growth factor (EGF), or nonconju-
gated EGF, on ice for 20 min to allow ligand-receptor binding
(
see
Note 6
).
3. Transfer cells to 37 °C for internalization for 5 min.
4. Acid wash (150 mM NaCl, 50 mM glycine, pH 3.0, ice-
chilled) cells to remove un-endocytosed EGF before incubat-
ing in complete DMEM pre-warmed to 37 °C for the chase.
5. Fix cells at various time points with 4 % paraformaldehyde.
Wash cells with 50 mM NH
4
Cl to quench autofl uorescence
caused by unreacted aldehydes. After rinsing with phosphate
buffered saline (PBS), permeabilize cells with 0.1 % saponin to
permit antibody access to antigen.
6. Label fi xed cells with primary antibodies for colocalization
analyses, followed by secondary antibodies with the desired
fl uorescence tag, in blocking buffer consisting of 5 % fetal
bovine serum (FBS) and 2 % BSA in PBS. To track the location
of EGF/EGFR complexes within the cell, primary antibodies
ʼ
3.2.1 Pulse-Chase
Experiments and
Compartment Localization
of Ligand-Bound EGFR
