Each experiment normally was performed in triplicate. RNA interference and gene overexpression studies A constitutively active form of Akt1 (CA-Akt1) and Mcl-1 cDNA (Upstate, Lake Placid, NY, USA) was generously provided by Dr Shengbing Huang (Mayo Clinic, Rochester, MN, USA) (Rahmani et al, 2003) for gene overexpression studies. Briefly, cDNA was cloned into pCDH1-MCS1-EF1-Puro vector (System Bioscience, Mountain View, CA, USA) for lentivirus packaging in 293 TN cells. Pancreatic cells were infected with lentivirus with multiplicity of infection (MOI) of 5 under selective Puromycin (1��gml?1). RNA interference was based on pGreenPuro system (System Bioscience) expressing small hairpin RNA (shRNA).
pGreen-FRS2��, pGreen-Mcl-1 and pGreenPuro-vec constructs, encoding shRNA for FRS2�� (sh-FRS2), Mcl-1 (shMcl-1) or a negative control (vector) respectively, were prepared by inserting the target sequence for human FRS2�� (shRNA1: 5��-CCGTGATAGACATCGAGAGAA-3�� or shRNA2: 5��-CCGTGCAGAAGAATTATTT-3��) or Mcl-1 (5��-GGACTTTTATACCTGTTAT-3��) into pGreenPuro. 293 TN cell was stably transfected with the constructs and three packaging plasmids using Lipofectamine 2000 reagent (Invitrogen) to package lentivirus; and then pancreatic cells were infected with lentivirus with multiplicity of infection of 5. Clones with stable downregulated FRS2�� or Mcl-1 expression were selected with puromycin (1��gml?1). Immunoblotting For immunoblot analysis, the cells were treated with the indicated agents and then collected in lysis buffer (Cell Signaling, Danvers, MA, USA).
Total protein was quantified using Coomassie protein assay reagent (Bio-Rad). An equal amount of protein (60��g) was separated by SDS�CPAGE and electrotransferred onto nitrocellulose membrane. The following primary antibodies were used: FGFR2, VEGFR1, p-VEGFR2 (Y1214) and VEGFR2, p-PDGFR�� (Y751) and PDGFR�� (1:1000, R&D Systems, Minneapolis, MN, USA); Mcl-1 (1:1000, BD PharMingen, Sparks, MD, USA); p-Akt(S473), Akt, p-Erk1/2(T202/T204), Erk, p-GSK3��(S9), GSK-3��, Bid, tBid, cyclin D1, cleaved caspase 3, cleaved poly(ADP-ribose) polymerase (PARP), human Bcl-2 and Bcl-xL (1:1000�C1:5000, Cell Signaling); p-FRS2��(Y196) and FRS2�� (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). ��-actin (1:500,000, Sigma) was measured as control for equal loading.
Blots were exposed to HRP-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:5000, KPL, Gaithersburg, MD, USA) and then developed by enhanced chemiluminescence (Pierce, Rockford, IL, USA). For semi-quantitative analysis, protein expression was quantified by densitometric analysis using Quantity One 4.6.5 (Bio-Rad). FRS2 phosphorylation ratio is calculated by the equation (p-FRS2��/FRS2��). AV-951 In Figure 2F, FGFR2 expression, and the phosphorylation status of FRS2, VEGFR2 and PDGFR�� were compared (��normalised’) to ��-actin of L3.