Journal: The Journal of Neuroscience
Article Title: Characterization of Densin-180, a New Brain-Specific Synaptic Protein of the O-Sialoglycoprotein Family
Figure Lengend Snippet: Densin-180 protein is enriched in PSD fractions, and its mRNA expression is brain-specific. A , Enrichment of densin-180 protein in detergent-extracted PSD fractions. Immunoblots were prepared with 50 μg ( lanes 1 , 2 ) of rat brain homogenate ( Hom ) and synaptosome fractions ( Syn ) and 7.5 μg ( lanes 3–6 ) each of synaptosome ( Syn ), once Triton X-100-extracted PSD ( 1T ), twice Triton X-100-extracted PSD ( 2T ), and once Triton X-100 and then sarcosyl-extracted PSD ( 1T + S ). Densin-180 protein band ( arrow ) is visualized with antibody M2 against densin-180. Molecular weight markers and position of the dye front ( open arrowhead ) are shown at left . B , Densin-180 Northern blot. Poly(A) + RNA (5 μg) from 13 different tissue samples was electrophoresed on a 1% agarose gel. The mRNA was transferred to Zeta-Probe blotting membrane (Bio-Rad), and all lanes were determined to have equal amounts of RNA by methylene blue staining. Blots were probed with a random prime-labeled PCR-amplified DNA fragment of densin-180 spanning nucleotides 1100–2170 (specific activity, 10 9 cpm/μg). A single broad band at 7.4 kb was detected ( large arrow ) on autoradiographs exposed for 14 d with an intensification screen. The blot was then stripped and reprobed with the 2 kb random prime-labeled human β-actin cDNA (specific activity, 10 7 cpm/μg). The autoradiograph of an 8 hr exposure with an intensification screen is shown in the bottom panel . The two forms of β-actin message are indicated ( small arrows ).
Article Snippet: The cDNA was prepared from mRNA with the First Strand cDNA kit purchased from Clontech (Palo Alto, CA).
Techniques: Expressing, Western Blot, Molecular Weight, Northern Blot, Agarose Gel Electrophoresis, Staining, Labeling, Polymerase Chain Reaction, Amplification, Activity Assay, Autoradiography