JCS02692 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig S1. Evaluation of Nd1-L mRNA stability in TLS-null cortical neuron. Primary cortical neurons were prepared from either wild-type (WT) or TLS-null (Null) mice. The neurons (12 DIV) were incubated in medium containing actinomycin D (5 mg/ml). Total RNA was harvested at the indicated time points (0, 1, 2, 3 hours). mRNA levels were measured by real-time PCR and normalized with respect to the amounts of 18S rRNA. The Nd1-L mRNA remaining at each time point is shown as a percentage of the RNA present in WT neuron at the time of the drug addition..

• Supplemental Figure 2 -

  Fig. S2. EMSA of Nd1-L 3¢-UTR region to identify the TLS binding sequence. P³²-labeled probes (~200-base in vitro synthesized riboprobes) were incubated with or without TLS. After in vitro binding, samples were run on non-denaturing gel. A TLS-dependent shift is observed at position 2700~3325 of the 3¢-UTR.

• Supplemental Figure 3 -

  Fig. S3. TLS binds rRNA. (Top) Western blot of mouse brain subfraction with anti-TLS antibody. Normally, TLS migrates as a 70 kDa protein in SDS-PAGE (lanes P1 and P2). The crude polysomal fraction (P3) shows a shifted band which is immunoreactive with anti-TLS antibody (shift). When P3 is UV-crosslinked to facilitate RNA binding to TLS, a TLS-positive band is supershifted to a higher molecular mass (supershift). (Bottom) Protein-RNA blot to show TLS association with rRNA (28S rRNA). S1, TLS-bound 28S rRNA in the cytoplasmic extract; P3, TLS-bound 28S rRNA in the crude polysomal fraction; P3 no IP, RNA extracts from crude polysomal fraction without IP by anti-TLS antibody. Each fraction was subjected to immunoprecipitation using anti-TLS monoclonal antibody (S1 and P3) or using IgG as a mock control (P3 no IP).