Investigating Signaling Processes in Membrane Trafficking - Membrane Trafficking - page 87

Fig. 3 Immunoprecipitated FLAG-Sec24A is phosphorylated by Akt. CHO-7 cells
were transfected with FLAG-Sec24 (isoform A), protein was immunoprecipitated
with FLAG antibody, and a kinase assay with or without Akt was performed.
Pellets were subjected to SDS-PAGE and Western blotting with phosphorylated
Akt substrates and FLAG antibodies

3.3 Western Blotting

1. Once the samples have suffi ciently separated ( see Note 16 ),
transfer to a nitrocellulose membrane using standard Western
blotting techniques.
2. Block the membrane in blocking solution with gentle rocking
for 1 h.
3. Rinse briefl y with TBST.
4. Incubate the membrane in primary antibody (phospho-Akt sub-
strates antibody) with gentle rocking overnight ( see Note 4 ).

5. Wash six times for 5 min in TBST with agitation ( see Note 4 ).
6. Incubate in secondary antibody with gentle rocking for 1 h.
7. Wash six times for 5 min in TBST with agitation.
8. Visualize labeling using ECL and LAS 500 imaging system.

An example of the results from this method can be found in
Fig. 3 .

1. Go to PhosphoSitePlus ( www.phosphosite.org ).
2. Search for your protein of interest.
3. Note the phosphorylation sites that have been found
( see Note 17 ).

3.4 Identifi cation
of Known
Phosphorylation Sites

We found that three known phosphorylation sites on DHCR24
affect its activity [ 15 ].

1. Go to scansite.mit.edu ( see Note 18 ).
2. Search for motifs within your protein of interest ( see Note 19 ).

3.5 Prediction
of Potential Kinases

We found that protein kinase C affects the activity of DHCR24
[ 15 ], not through a known phosphorylation site, but Scansite pre-
dicts eight potential PKC phosphorylation sites, two of which are
considered “medium stringency.”