Journal: EMBO Reports
Article Title: The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development
Figure Lengend Snippet: The micro RNA ‐generating complex localizes at the neuronal endoplasmic reticulum Components of the RLC co‐precipitate with ER markers in density gradient fractionation. The postnuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers, calnexin, ribophorin I, and Climp63, co‐sedimented with ribosomes (RPS6, ribosomal protein subunit 6 ), mitochondria (Tim23), and RLC proteins at fractions 3–5. The bottom graph shows the density of each fraction. TRBP, PACT, and Ago2 partially co‐localize with the ER marker mCherry‐CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry‐CLIMP63 were immunostained with TRBP, PACT, or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10 μm). GFP‐Dicer partially co‐localizes with mCherry‐CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150 nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2 μm or 10 μm). The majority of Dicer, TRBP, and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (** P
Article Snippet: Nuclei were pelleted by centrifugation 700 × g , 10 min, and the postnuclear supernatant (PNS) was mixed with 50% Optiprep density gradient medium (D1556, Sigma) to make a 35% solution and loaded underneath a 2.5–30% continuous Optiprep gradient.
Techniques: Fractionation, Marker, Expressing, Fluorescence, Microscopy