Figure 3.

Activation of AMPK at the early stage of differentiation is positively regulated by IGFBP-2 and requires IGF-I receptor activation. A, Cell lysates obtained from LacZ- or IGFBP-2-overexpressing cells on indicated day after exposure to DM were immunoblotted with anti-pAMPK (T172), IGFBP-2 or β-actin. B, Lysates from MC-3T3 cells expressing shRNA sequence targeting LacZ (Ctrl Si) or IGFBP-2 (IGFBP-2 Si) obtained on the indicated day after DM exposure were immunoblotted with anti-pAMPK (T172), IGFBP-2, or β-actin. C, Lysates from MC-3T3 cells expressing shRNA sequence targeting IGFBP-2 (IGFBP-2 Si) following a 3-day DM exposure in the absence or the presence of IGF-I (100 ng/mL) alone or IGFBP-2 (1 ug/mL) alone or both or IGF-I and IGFBP-2 plus antifibronectin antibody-3 (anti-Fn) (500 ng/mL) were immunoblotted with anti-pAMPK (T172) or anti-AMPK. Each bar is the ratio of the scan value of the pT172AMPK band divided by the AMPK band. **, P < .01 indicates significant differences between two treatments. D, Lysates from MC-3T3 cells obtained on the indicated day after DM exposure were immunoblotted with anti-IGFBP-2. E, Lysates from IGFBP-2 overexpressing cells obtained on day 3 after DM exposure in the absence or the presence of PQ401 (25uM) were immunoblotted with anti-pAMPK (T172) or anti-AMPK.