MEFS, U2OS or HEK293 cells were grown in 10% FBS (v/v), L-glutamine and Pen/Strep. Cells were washed twice with Earles balanced salt solution (EBSS) and then either incubated in EBSS for 1h or replenished with full media plus 50 ng/ml human IGF1 (1 hour also). Cells were then washed with PBS and lysed in 0.5% NP-40 lysis buffer. Lysis were immunoblotted with either anti-UVRAG at 1 ug/ml, anti-4E-BP1 (cell signalling 9452) or anti-Tubulin (Calbiochem CP06)

Anti-UVRAG S323D can immunoprecipitate UVRAG from mouse and human cells. And it can also co-immunoprecipitate Beclin. UVRAG was immunopreipitated from MEF or HEK293 cells using 1-3 ug of anti- UVRAG per mg of lysate. The IPs were blotted with anti-UVRAG (MBL M160-3) or anti-Beclin (MBL PD017)