Zhang, et al (2001): Figure 2

Supplementary Material From Singer Lab Publications:

Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility

Neuron 31(2):261-275 (2001 August 2)

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Figure 2. ZBP1 Forms an RNP Complex with the ß-Actin Zipcode in Cultured Neurons

(A) A specific RNA-protein complex was observed with the 54 nt zipcode, but not with the 54 nt mutated zipcode (lanes 1 and 2). Formation of RNA-protein complexes with the ß-actin zipcode was significantly inhibited by increasing concentration (from 1 to 5 pmol) of the antisense oligonucleotides, but not by the control oligonucleotides.

(B) Expression of ZBP1 in cultured forebrain neurons shown by RT-PCR and sequence analysis (within the amplified 486 nt sequence, red). RBD, RNA binding domain. NLS, nuclear localization sequence. KH, domain showing homology to hnRNP K.

(C) Western Blot analysis of chick forebrain cultures using anti-ZBP1 (VgRBP) antibody (Zhang et al., 1999c) showed a single band at approximately 66 kDa.

(D) UV crosslinking of neuronal culture extracts identified a protein of approximately 66 kDa that was bound to the 32P-labeled zipcode.

(E) Immunodepletion of neuronal culture extracts with anti-ZBP1(VgRBP) antibody significantly reduced the formation of the RNA-protein complex with ß-actin zipcode as in (D).