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Figure 2 | BMC Cancer

Figure 2

From: Targeting both IGF-1R and mTOR synergistically inhibits growth of renal cell carcinoma in vitro

Figure 2

IGF-1R expression. (A) ACHN cells were plated overnight in 6-well plates as described in Materials and Methods. Cells were than exposed to hR1, Hex-hR1, or control hRS7 antibodies before being lysed and 20 μg protein from these lysates subjected to SDS-PAGE (4-20%) followed by blotting with an anti-IGF-1Rβ antibody. IGF-1R time-course down-regulation after exposure to constant amount of either hR1 or Hex-hR1. (B) Cells were exposed to indicated amounts of hR1, Hex-hR1, or hRS7 (anti-Trop-2 antibody) for 6 h before being lysed for Western blotting. β-actin served at the loading control. Blot shown is representative of three repeat experiments. (C) Ratio of IGF-1R to β-actin loading control normalized to untreated cells for the various doses of hR1, Hex-hR1 or control hRS7. Data are shown as mean ± standard deviation. Significance set at P < 0.05 for paired t-test of three experiments. (D) Indicated cells were lysed subjected to immunoprecipitation to determine IGF-1R/IR hybrid receptors, as described in Materials and Methods. A 20-μL aliquot from each immounoprecipitation preparation was subjected to gel eletrophoresis and Western blotting. Hep G2 served as the positive control cell line in these experiments [22]. Blot shown is representative of two repeat experiments. To confirm presence of IGF-1R in IP samples, IP blots were probed with anti-IGF-1R antibody. Additionally, cell lysates were subjected to Western analysis and probed with an anti-IRβ antibody to show relative levels of IR in the various cell lines. β-actin served at the loading control.

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