Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability
Figure Lengend Snippet: ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p<0.05; **p<0.01, compared to control. DOI: http://dx.doi.org/10.7554/eLife.17304.003
Article Snippet: 20–30 μg of protein extracts were loaded into SDS-PAGE gels, transferred to nitrocellulose membranes, and western blotting performed using lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:1000) to represent the cytosolic fraction, Lamin-B1 (Abcam Cat# ab16048 RRID: AB_443298 , 1:1000) representing the nuclear fraction, Serca2a (1:1000, Dr. Periasamy, Ohio State University) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) to observe localization.
Techniques: Microarray, Expressing, Hybridization, Sequencing, Transfection, Staining