Anti pan p85

Cell lysate samples were denatured and reduced in 1 × NuPAGE LDS Sample Buffer (NP0007, Invitrogen) with 20 mM DTT at 70°C for 10 min. Lysate, AP and IP samples were subjected to SDS-PAGE on NuPAGE 4–12% Bis-Tris Gels (Invitrogen) and proteins transferred onto PVDF membranes using the XCell SureLock Mini-Cell Electrophoresis System and XCell II Blot Module (Invitrogen) according to the manufacturer's instructions. Membranes were blocked in 5% BSA (w/v) TBS-Tween and incubated with the following primary antibodies overnight at 4°C: anti-p110δ (sc-7176, Santa Cruz), anti-pan-p85 (06-195, Millipore), anti-phosphotyrosine (05-321, Millipore), anti-BCAP (generous gift from T. Kurosaki), anti-pAKT T308 (4056, Cell Signaling Technology), anti-AKT1 (2967, Cell Signaling Technology); all in 5% BSA TBST. Washed membranes were incubated with the following secondary antibodies for 1 h at room temperature: goat anti-rabbit IRDye® 680RD (926-68071, LI-COR) or goat anti-mouse IRDye® 800CW (926-68070, LI-COR); in 5% BSA-TBST. For detection of pAKT and AKT1, membranes were incubated with both primary or secondary antibodies simultaneously. Membranes were imaged using an Odyssey® CLx Imaging System (LI-COR) and images were quantified using Image Studio Lite Version 5.2 (LI-COR). Statistical analyses as detailed in figure legends were performed in GraphPad Prism (GraphPad Software, Inc.).