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Fig. 6. Binding to hnRNP K and expression of NF-M mRNA in vivo. (A,B) Co-immunoprecipitation and RT-PCR of NF-M (A) and peripherin (B) mRNAs with hnRNP K from juvenile brain. (1) NF-M PCR from plasmid template with Xenopus NF-M cDNA insert, which served as a positive control for NF-M PCR amplification. (2) NF-M RT-PCR of sample prior to co-IP, demonstrating NF-M mRNA presence in the lysate. (3) NF-M RT-PCR of anti-hnRNP K co-IP. (4) NF-M RT-PCR of anti-β-galactosidase co-IP, a control for non-specific IP. (5) EF1-α RT-PCR of hnRNP K co-IP, demonstrating absence of mRNAs not associating with hnRNP K. (6) EF1-α RT-PCR of TIC, demonstrating its presence in lysate. (B) (1,2) peripherin PCR from plasmid template, which served as positive controls for peripherin PCR with each primer set. (3) peripherin RT-PCR of lysate prior to co-IP using the first pair of primers (30 cycles). (4,5) peripherin RT-PCR of anti-hnRNP K co-IP with the first (30 cycles) and second (15 additional cycles) pair of nested primers, respectively. Std, 1 kb DNA ladder (NE Biolabs). (C-E) Expression of NF-M and peripherin mRNAs in unilaterally injected hnRNP K MO animals. Dorsal views in whole mount of the entire animal (C), spinal cord (C1) and head (C2) of a stage 39/40 tadpole, processed for NF-M in situ hybridization (digoxigenin-alkaline phosphatase). Stained neurons are on both sides of the spinal cord (C; arrowheads in C1) as well as in brain, trigeminal ganglion (Vth) and retinal ganglion cells (Rgc, C2). (D) Horizontal confocal optical section of a unilaterally injected stage 39/40 hnRNP K MO tadpole processed for NF-M FISH. Rostral is towards the upper left. (E) Dorsal view of the head of a stage 39/40 hnRNP K MO tadpole stained for peripherin mRNA. R, rhombomeres. |
