Figure 2.

The SCL protein isoform ratio is modulated by translation initiation factor activity. (A) HA-tagged SCL was cotransfected with PKRΔ6 (to activate eIF2α), eIF4E, or control empty vector (−) into HEK293A. SCL expression was analyzed by immunoblotting using a HA-epitope-specific antiserum. The schematic representations of the SCL mRNA on the left indicate SCL initiation sites in relation to the protein bands. An alternative CUG initiation codon is depicted with an arrowhead (▹). (B) HD3 erythroblasts were infected with retrovirus encoding PKRΔ6, eIF4E, or a control vector (−) and endogenous SCL was examined by immunoblotting using chicken SCL-C terminus-specific antiserum. (C) HEL cells were infected with PKRΔ6 or control virus (−) and endogenous SCL expression was examined by immunoblotting using human SCL-C terminus-specific antiserum. (Right) Expression of eIF4E transgene was controlled by immunoblotting. eIF2α-specific and eIF2α phosphorylation-specific antibodies (eIF2α-P) were used to determine the effect of PKRΔ6 on eIF2α. Because the antibody raised against human eIF2α fails to detect the chicken homolog, a nonspecific band (*) detected by the eIF2α-P antibody serves as an internal control for the loading of HD3 extracts. Northern blots of polyA⁺ RNA revealed no alterations in scl RNA splicing pattern between control (−) and cells expressing PKRΔ6 or eIF2α.