Journal: Nucleic Acids Research
Article Title: Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5? untranslated region
Figure Lengend Snippet: BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is γ-ATP labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
Article Snippet: A [γ-32 P]-ATP labeled 50 bp DNA ladder (New England Biolabs, Beverly, MA, USA) was used as a size reference on the gel.
Techniques: Variant Assay, Agarose Gel Electrophoresis, Rnase Protection Assay, Incubation, Labeling, Marker, Real-time Polymerase Chain Reaction, Expressing