1. The first evidence that PI3K gets activated by tyrosine kinases came from studies on the soluble src kinase and the platelet-derived growth factor receptor (9,10). Although the lysis buffer used in these prior studies works well for the precipitation of either of these kinases, it is not optimal for the immunoprecipitation of IRS-asso-ciated PI3K (Whiteman and Birnbaum, unpublished observation). This is perhaps the result of an association of IRS proteins with the cytoskeleton following hormonal activation. Figure 3 compares a conventional buffer (20 mM Tris-HCl [pH 7.5] containing 137 mMNaCl, 1 mMMgC^, 1 mMCaC^, and 100 \\MNa3VO4, 1% igepal [Sigma], and 10% glycerol) used for PI3K assays (11) with the one described herein.

2. One can mix the 4G10 and PY20 monoclonal anti-phosphotyrosine antibodies and use this cocktail in the immunoprecipitation.

3. Phosphatidylinositol is commonly used as a substrate in the PI3K assay. The lipid is inexpensive and easy to store, and its phosphorylated product, PI3P, is easily extracted from aqueous solutions and separated from ATP by TLC. However, the lipid can be a substrate for PI4- and PI5-kinases, and the lipid products produced by the phosphorylation of PI by these kinases, PI4P and PI5P, cannot be separated from PI3P using the TLC solvent system described herein. Thus, without the use of PI3K inhibitors, such as wortmannin or LY29002, one cannot confirm that the kinase being assayed is PI3K and not another PI-kinase. An alternative is to use PI(4,5)P2 (Sigma), which can only be phosphorylated by PI3K. The charge makes it insoluble in chloroform and its product is harder to extract quantitatively from

Fig. 3. Sample TLC plate showing the formation of PI(3)P produced by PI3K. PI3K activity was immunoprecipitated from 3T3-L1 adipocytes treated with or without insulin as described in the text. Buffer A is the buffer described herein, whereas buffer B is the conventional buffer described in Note 1.

aqueous solutions and more difficult to separate from ATP by TLC. Also, this lipid does not form vesicles following sonication in TE buffer. To store PI(4,5)P2, resuspend it in a chloroform-methanol-water (5:10:2, v/v/v) solution. To suspend in vesicles, mix PI(4,5)P2 1: 1 with another carrier lipid, such as phosphatidylserine. Some investigators use equal amounts of all three phosphoinositide substrates [i.e., PI, PI(4)P, PI(4,5)P2] plus phosphatidylserine, which will lead to the formation of three different products after the PI3K assay. See Note 5 regarding differences in TLC solvent systems if using PI(4,5)P2 as a substrate.

4. The dried lipids will form a visible film on the bottom of the microcentrifuge tube. During sonication the solution will initially turn cloudy and then clear as lipid-vesi-cles form. The film on the bottom of the tube will noticeably disappear.

5. If using PI(4,5)P2 as a substrate, prepare a solvent system composed of 1-pro-panol/acetic acid-water (65 :4.1:30.9, v/v/v) (12).

6. In 3T3-L1 adipocytes, insulin-stimulated PI3K activity is maximal within 1 min and remains elevated for hours (13). By contrast, some growth factors, such as platelet-derived growth factor, cause a more transient increase in PI3K activity (13). We recommend 1-5 min, because this is the peak time for both insulin and other growth factors. For other agonists, this must be determined empirically.

7. Although the amount of protein-A-agarose used in the immunoprecipitation here is sufficient to precipitate the entire amount of primary antibody in the lysate, some researchers have difficulty seeing this relatively small amount. This increases the likelihood of accidentally aspirating some of the beads. One can either add excess agarose conjugated to protein-A or anti-mouse antibodies, or, as an less expensive, add unconjugated agarose to help with visualizing the immunoprecipitate.

8. The wash with high concentrations of lithium is included to inhibit inositol phosphatases (14).

9. The aqueous phase contains most of the free [32P]ATP, which will show up as a large origin on the TLC plate. This is not problematic for analysis but is not as aesthetically pleasing, particularly if the size of the ATP spot at the origin varies greatly between samples. To extract the lower phase, depress the plunger of a p100 Pipet-man (Rainin, Emeryville, CA) almost all of the way and then move the tip through the upper aqueous phase and the layer of agarose beads at the interphase. Depress the plunger the rest of the way to eject the drop of aqueous phase that entered the tip as it moved through the upper layer. Slowly release the plunger to draw the lower phase into the Pipetman and transfer it to a new microfuge tube. Extract 20-30 iL each time, and repeat until the lower phase is completely removed.

10. The TLC plate can also be saturated with potassium oxalate by allowing the oxalate solution to travel up the plate in a TLC developing tank. The oxalate solution can also be made in pure methanol, which will shorten the drying time.

11. The final lipid products are easy to identify. For PI(3)P, it is the highest migrating phosphorylated species. One can, however, load lipid standards that are available commercially from Avanti Polar Lipids (Alabaster, AL) and Echelon Research Labs (Salt Lake City, UT) on adjacent lanes. To detect these standards, place the TLC plate in a sealed chamber with iodine and allow the lipids to be stained by the iodine vapor.

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