Northern Blot Analysis of Pabpn1 Expression

Total RNA from tissues and cells was isolated as described above under, ‘RNA preparation, cDNA and qRT-PCR.’ RNA (20 μg) was loaded on a 1.4% agarose denaturing formaldehyde gel. RNA was transferred to Amersham Hybond-N+ membrane (GE Healthcare and Life Sciences) with 10× SSC buffer. RNA was UV-crosslinked and prehybridized at 65°C in 100 μg/ml salmon sperm DNA (Invitrogen) in Rapid-Hyb buffer (GE Healthcare Life Sciences). PCR using primers spanning exons 2–3 (forward primer) and exons 6–7 (reverse primer) generated an ∼400 nucleotide Pabpn1 probe (22 (link)). This Pabpn1 PCR product was [α-³²P]dCTP (Perkin Elmer) labeled using the Amersham RediPrime II DNA Labelling System (GE Healthcare Life Sciences) and purified with illustra MicroSpin G-25 columns (GE Healthcare Life Sciences). The Pabpn1 probe was hybridized to the membrane rotating at 65°C overnight. The reverse 18S oligo was end-labeled with [γ-³²P]dATP (Perkin Elmer) using PNK (New England Biolabs) and purified with illustra MicroSpin G-25 columns (GE Healthcare Life Sciences). The 18S probe was hybridized rotating at 42°C overnight. A Typhoon phosphorimager or autoradiography film were used to detect labeled probe. The rRNA 18S films were exposed for 1 h while Pabpn1 films were exposed for ∼50 h. Northern blots were quantified using ImageJ software. The band for 18S rRNA was used to normalize RNA loading.