FIGURE 1:

LanCL2 is necessary for maximal Akt phosphorylation. All experiments were performed in HepG2 cells unless otherwise indicated, with cell lysates subjected to Western analysis. (A) Cells were infected with lentiviruses expressing LanCL2 shRNA (“L”) or a hairpin of scrambled sequence as control (“C”) for 1 d; this was followed by puromycin selection for 2 d. The cells were then either maintained in growth medium (“growth”), or serum starved overnight (“starved”) and restimulated with 10% FBS for 20 min (“stimulated”). (B) Cells were infected as in A and maintained in growth medium before lysis. (C) Cells were infected with lentiviruses expressing LanCL2 shRNAs (“L-1” and “L-2”), serum starved overnight, and then stimulated with 30 nM insulin for 20 min. (D) Cells were transfected with FLAG-tagged LanCL2 together with a plasmid carrying a puromycin-resistant gene and selected in puromycin for 3 d. (E) Three cell lines as indicated were treated as in A, serum starved overnight, and then stimulated with 10% FBS for 20 min. (F) Cells were infected with lentivirus expressing LanCL1 shRNA for 1 d; this was followed by puromycin selection for 2 d. The cells were then serum starved overnight and restimulated with 10% FBS for 20 min.