Journal: PLoS ONE
Article Title: Identification of RNF213 as a Susceptibility Gene for Moyamoya Disease and Its Possible Role in Vascular Development
doi: 10.1371/journal.pone.0022542
Figure Lengend Snippet: ( A ) RNF213 mRNA expression. Total RNA from the indicated human tissues was reverse-transcribed to cDNA, and real-time quantitative PCR was performed. ( B ) RNF213 protein expression. LCL, HUVEC, and CASMC were lysed and immunoblotted using an anti-RNF213 antibody. HEK293 cells transiently expressing RNF213-HA (+) or control cells (−) were immunoblotted using anti-RNF213 and anti-HA antibodies. ( C ) Subcellular localization of RNF213. HeLa cells transiently expressing RNF213-HA were stained with an anti-HA antibody. ( D ) Self-ubiquitination of RNF213. HEK293 cells transiently expressing RNF213-HA and Myc-ubiquitin (Myc-ub) were lysed and subjected to immunoprecipitation (IP) using an anti-HA antibody, followed by immunoblotting (IB) using an anti-Myc antibody. As a control, immunoblotting was also performed with an anti-HA antibody. ( E ) ATPase activity of RNF213. Free phosphate released from ATP by the ATPase activity of a recombinant RNF213 fragment (a.a. 2359–2613) tagged with GST was measured using the Malachite Green method. a.a., amino acid. IP, immunoprecipitation. IB, immunoblot.
Article Snippet: A human adult normal tissue mRNA northern blot I (Biochain) was probed in accordance with the supplier's recommendations.
Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Staining, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Activity Assay, Recombinant