Changes in TIMP3 protein expression relate to miR-21 expression.

<p>Cells were collected 24 hours post-transfection for lysis, total RNA isolation, and flow cytometry. Twenty micrograms of protein were loaded and subjected to immunoblotting for the miR-21 putative target proteins TM1, TIMP3, PDCD4, and PTEN (<b>A</b>). Immunobloting for β-actin was used as the loading control. The RNA was converted to cDNA and Real‑Time PCR for TIMP3 was performed (<b>B</b>). Induction of expression was calculated relative to β-actin and compared to control-transfected cells. Cells were incubated with PE-anti-TIMP3 or isotype control Ab, washed and fixed in 1% formalin. A total of 10,000 cells were analyzed on a LSRII flow cytometer <b>(C)</b>. Error bars represent standard error.