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. 2008 Apr 11;283(15):9787–9796. doi: 10.1074/jbc.M708839200

FIGURE 4.

FIGURE 4.

Identification of TBC1D1 as PAS-160 in TA muscle. A, to determine whether AS160 was the phospho-Akt-substrate detected at a molecular weight of 160 (PAS-160) in soleus and tibialis anterior muscle, AS160 was immunoprecipitated from insulin-stimulated soleus and tibialis anterior muscle lysates. Lysates, supernatants, and immunoprecipitated AS160 were immunoblotted (IB) for both AS160 and PAS-160. Gels for AS160 and PAS-160 immunoblots were loaded identically with volume equivalents of lysates and supernatants and a supra-proportional amount of AS160 immunoprecipitate to compensate for inefficient elution. AS160 and PAS-160 immunodepletion and immunoprecipitation patterns matched in soleus but not tibialis anterior muscle. B, to determine the identity of PAS-160 in tibialis anterior muscle, lysates were first immunodepleted of AS160; then the PAS antibody was used to immunoprecipitate the remaining PAS-160. AS160 and PAS-160 immunoprecipitates were subjected to SDS-PAGE and stained with Coomassie Blue. The AS160 and PAS-160 bands were excised from the gel and identified by mass spectrometry. The identity of AS160 was confirmed, and PAS-160 was identified as TBC1D1.