Figure 3 | Scientific Reports

Figure 3

From: CREB1 is affected by the microRNAs miR-22-3p, miR-26a-5p, miR-27a-3p, and miR-221-3p and correlates with adverse clinicopathological features in renal cell carcinoma

Figure 3

miTRAP analyzing 3′-UTR of CREB1. (A) Scheme illustrating the miTRAP method using 3′-UTR of CREB1. (B) Western blot-based detection of indicated proteins isolated from the miTRAP input sample (MZ2733RC) or co-precipitated with the used resin (amylose only), 2x MS2 loop RNA (MS2 loop only) or the different parts of the 3′-UTR of CREB1, respectively. Same blot was re-probed for β-Actin serving as negative control to exclude unspecific binding to the different RNAs. Detection of maltose binding protein (MBP) on the same blot ensures equal loading of the resin. Uncropped blot is shown in Supplementary Figure 4. (C) Co-purification of candidate miRNAs after the miTRAP procedure using CREB1 3′-UTR or MS2 loop control analyzed by qRT-PCR. Values are normalized to the input expression. miR-222–3p served as negative control (part 1) while miR-17–5p (part 2) was used as positive control.

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