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. 2018 Apr 20;9(30):21444–21458. doi: 10.18632/oncotarget.25118

Figure 2. MAP3K1 depletion correlates with increased IRS1 stability.

Figure 2

(A) MCF7 parental, CR1.4 and CR2.5 cells were treated with 250 nM AZD8835, 250 nM AZD5363 or DMSO 24h prior to cell lysis. Cell lysates were collected and immunoblotted with indicated Abs. (B) MCF7 parental, CR1.4 and CR2.5 cells were incubated with 50μg/ml cycloheximide (CHX) at the indicated time points in the presence or absence of AZD5363 and immunoblotted with total- and phospho (Ser312)-IRS1 specific Abs. Densitometry quantification is shown in Supplementary Figure 2D. (C) Whole cell lysates were incubated with PI3K regulatory subunit (p85) Abs overnight. Immuno-complex pull-downs were performed using magnetic Protein A beads and immunoblotted with the indicated Abs. (D) Immunoblots from MCF7 parental and CR2.5 cells cultured in the presence (+) or absence (−) of AZD5363 and immunoblotted with the indicated Abs.