Nelfe

Hep3B or Huh1 HCC cells were cultured on 15 cm plates until 80% confluent. Cells were then washed 2× with ice cold 1× PBS on ice. 1mL of RIPA buffer supplemented with 1mM of PMSF was then added onto plates and incubated on ice for 10 min. Cells were then scraped and collected followed by a 10 min incubation on ice. Lysates were centrifuge for 10 min at 13K RPM for 10 min at 4C. The supernatant was then transferred into a new microcentrifuge tube. For IP, 50 μl of neutravidin slurry (Thermo Scientific) were washed twice with 1× PBS. The slurry was then centrifuged for 1 min at 1500 RPM and the supernatant was removed via pipetting. 8 μl of 100 μM mRNA oligos (HPLC grade generated by IDT) were then incubated with the slurry and 100ul of 1× TBS for 2 hr, washed 3× with 1×TBS and incubated with 150ul of whole lysates freshly prepared and incubated overnight in 4 °C with rotation. 4 μl (~2.5%) of the lysates were used as input. The slurry was then washed and immuno-blotting was performed to test for binding (NELFE from Abcam (cat. ab170104)). For oligo sequence, see Supplemental Materials.