Cells were lysed as reported previously.58 (link) Total proteins were extracted from AML cell lines. Equal amount of protein extract (60 μg) was transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h with 5% milk (Sigma-Aldrich) at room temperature, and then incubated with primary antibodies directed toward Lin28A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (Merck Millipore, Billerica, MA, USA) and β-actin (Sigma-Aldrich), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Protein bands were visualized and quantified as described previously.59 (link)
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