³⁵ S Met/Cys label Fao cells

100mm dish:

1.deprive cells overnight in RPMI containing 2% FBS

2. wash cells 3 times in 5 ml PBS

3. grow cells for 1 hour in 5 ml of RPMI without Met/Cys (Sigma, R7513) supplemented with 2mM Glu

4. change the medium to 4 ml RPMI without Met/Cys supplemented with 2mM Glu and 200mCi ³⁵ S Met/Cys (ICN, Trans ³⁵S-label #51006)

5. wash cells 5 times with 5 ml RPMI without Met/Cys and grow 15 minutes in 4 ml RPMI without Met/Cys supplemented with 2mM Glu/0.1% BSA

6. add insulin for indicated times

7. lyse cells in 600ml lysis buffer, immunoprecipitate with 5ml of aIRS-2 (#110) overnight on ice

8. separate proteins by 7% SDS-PAGE and dry the gel for 1.5 hours

9. expose overnight