Figure 9
From: Nck1 depletion induces activation of the PI3K/Akt pathway by attenuating PTP1B protein expression
Nck1 depletion affects neither PTP1B gene transcription nor protein stability. (A) Quantitative RT-PCR analysis of PTP1B mRNA levels in HepG2 cells transfected with control or Nck1 siRNA. Data are presented as mean ± SEM from five independent experiments. The expression levels of PTP1B mRNA in control siRNA-transfected cells were set to one. (B) Quantitative RT-PCR analysis of PTP1B mRNA levels in liver of Nck1+/+ and Nck1+/+ mice. Data are presented as mean ± SEM of four mice under each genotype. The expression levels of PTP1B mRNA in Nck1+/+ mice were set to one. (C) HepG2 cells transfected with control or Nck1 siRNA were treated with 10 μm of the PI3K inhibitor LY294002 or 10 μm of the Akt inhibitor Akti 1/2 for 24 h before harvesting. Equal amount of proteins from total cell lysates were subjected to immunoblot with indicated antibodies. Data are representative of three independent experiments performed in duplicate and showing similar results. (D) Twelve hours after transfection, HepG2 cells transfected with control or Nck1 siRNA were treated with 100 μg/ml of cycloheximide (CHX) for additional 12, 24 or 36 h. Equal amount of proteins from total cell lysates were subjected to immunoblot with indicated antibodies. The graph represents PTP1B levels at different times of CHX exposure as mean ± SEM from three independent experiments with PTP1B levels at time 0 in control and Nck1 siRNA-transfected cells fixed to one. (E) HepG2 cells transfected with control or Nck1 siRNA were treated with 0, 10, 25, 50 and 100 μm MG132 for 6 h. Equal amount of proteins from total cell lysates were subjected to immunoblot with indicated antibodies. Data are representative of three independent experiments showing similar results. (F) HepG2 cells transfected with control or Nck1 siRNA were treated with 100 μm chloroquine (CQ) for 12 or 24 h. Equal amount of proteins from total cell lysates were subjected to immunoblot with indicated antibodies. Data are representative of three independent experiments showing similar results.