(a) Western blot of total protein lysates prepared from MDA-MB-231 cell pools as described in Figure 1a was probed for phosphorylated IκBα protein (Ser 32; Cell Signaling Technology, Danvers, MA, USA), stripped and reprobed for total IκBα protein (top panel). The ratio of phosphorylated band intensity to total band intensity was normalized as in Figure 1b and shown below the western panel. Western blot in the lower panel shows that levels of NF-κB (65 kDa) are equal in control cells and miR-146a/b-expressing cells relative to β-actin. (b) EMSA for binding to a consensus NF-κB DNA oligo probe using control (C), miR-146a (146a) and miR-146b (146b) nuclear extracts loaded in duplicate lanes (left panel). The position of the gel origin, NF-κB gel shifted band and free probe are indicated. Right panel shows specificity of NF-κB DNA binding as antibodies to the p65 and p50 subunits of NF-κB supershifted the NF-κB band (p65 Ab and p50 Ab lanes) whereas a control antibody (con Ab, an estrogen receptor antibody) had no effect. Bottom panel: western blot of nuclear extracts used for EMSA probed for HDAC2 (Santa Cruz Biotechnology) shows equal protein content of the extracts. HDAC2, histone deacetylase 2; miR, microRNA; NF-κB, nuclear factor-κB; EMSA, electrophoretic gel shift assays.