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. Author manuscript; available in PMC: 2017 Apr 15.
Published in final edited form as: Mol Cell Endocrinol. 2015 Dec 28;425:48–60. doi: 10.1016/j.mce.2015.12.006

Figure 7. The effects of CK2 and PKC inhibition on IGF-1R autophosphorylation.

Figure 7

HepG2 cells were treated in leucine plus (450 μM) or leucine deprived (0 μM) media with BIS or with TBB, a pharmacological CK2 inhibitor, for 24 hours (n=3). HepG2 cell media samples were aliquoted to contain equal concentrations of IGFBP-1 and buffer-exchanged to serum-free P6 media (DMEM/F12 with pyruvate). Aliquots were then incubated with human recombinant IGFI (25 ng/mL) for 2 hours to allow IGFBP-1 binding to IGF-I, followed by a 10 minute exposure to P6 cells to allow induction of IGF-I-mediated IGF-1Rβ autophosphorylation (Tyr1135). A representative western immunoblot of post-treatment P6 cell lysates (50 μg per lane) assessed for IGF-IR autophosphorylation (Tyr1135). Leucine deprivation-stimulated IGFBP-1 phosphorylation reduced IGF-1R activation; leucine deprivation was unable to elicit this effect in the presence of BIS or TBB. Values are displayed as mean + SEM. *p< 0.05, **p= 0.001-0.05, ***p < 0.0001 versus control; One-way analysis of variance; Dunnet’s Multiple Comparison Test; n=3. – IGF-I: Negative control, no IGF-I, no IGFBP-1. +IGF-I: Positive control, 25 ng/mL IGF-I, no IGFBP-1. C:450: Control, 450 μM leucine. C:0: Leucine deprivation, 0 μM leucine. TBB:450: TBB (1 μM), 450 μM leucine. TBB:0: TBB (1 μM), 0 μM leucine. BIS:450: Bisindolylmaleimide (7.5 μM), 450 μM leucine. BIS:0: Bisindolylmaleimide (7.5 μM), 0 μM leucine.