Am2294

CLIP was performed as described (41 (link)) with the following modifications. Briefly, two 15 cm dishes of HEK293T cells per CLIP were transfected with pEGFPC1-hLa (42 (link)), or indicated GFP-hLa variants or myc-tagged pcDNA-PABPC1 (43 (link)) (PolyJet, SignaGen). Twenty four hours post-transfection, cells were UV-crosslinked (Stratalinker at 254 nm, 1000 mJ) and cells were lysed in RIPA buffer (10 mM Tris–HCl pH8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF). Lysates were then treated with 3 μl of 1:100 RNAse I (100 U/μl AM2294 Invitrogen) or 40 μl of a 1:100 dilution of RNase T1 (1 U/μl AM2283 Ambion) and 2 μl DNase I (2 U/μl AM2238 Ambion) at 37°C for 3 min. Antibodies used were anti-cmyc (Abcam ab21060) or anti-GFP (Abcam ab1218). coIPs were performed using Protein G magnetic Dynabeads (Invitrogen). Antibodies were incubated with Dynabeads in 150 μl RIPA buffer for 1 h at room temperature, then antibodies/Dynabeads were incubated with lysates for 2 h at 4°C. Complexes were washed 2× with RIPA buffer and 3× with proteinase K buffer prior to digestion with 10 μl proteinase K (buffer and enzyme supplied by Invitrogen, #100005393 20 mg/ml). Eluted RNA was probed with ³²P radiolabeled dT (40 (link)), stripped and reprobed for pre-tRNA Met-e (probe sequence AAA TTA TTG TGC CCC G) via northern blot.