Effects of Pan3 isoforms and their domain truncations on Pan2 deadenylase activity in vivo. NIH3T3 cells were transfected with a plasmid coding for HA-Pan2 alone or together with a plasmid coding for one of the HA-tagged WT or truncated Pan3 isoforms. β-Globin mRNA constitutively expressed ( A ) or transiently induced ( B – D ) in the same cells served as a reporter transcript. Total cytoplasmic RNA was extracted for Northern blot analysis to check poly(A) size distribution of the β-globin reporter mRNA ( upper panels of A , B ; upper left panels of C , D ). Poly(A) – RNA samples were prepared with oligo(dT) and RNase H treatment. Levels of the indicated HA-tagged proteins were visualized by Western blot analysis ( lower panels of A , B ; lower left panels of C , D ). The poly(A) size distribution profiles ( middle panels of A , B ; right panels of C , D ) were obtained by scanning each lane of the Northern blots using ImageJ software. Red arrows ( A , B ) highlight changes of the poly(A) size distribution profile. Red and blue dashed lines ( C , D ) serve as a reference to compare the effects caused by different Pan3 domain truncations on the poly(A) size distribution.