Figure 3. Western analysis of LIN-14 isoform temporal regulation. Approximately 2 ug of total protein was loaded in each lane. (A) The LIN-14 isoforms do not display stage- specific expression differences. Both exon-specific antibodies detect a steady decrease of protein from hatching through the L1 larval stage. Animals were staged as described in materials and methods, and the L1 molt occurs at 15 hr of postembryonic development. Note the doublet observed with anti-LIN- 14B, which may represent the two LIN-14B splice variants, B1 and B2. (B)
lin-14(
n360) and
lin-14(
n355n534) are null mu- tants for the expression of the LIN-14B1 and B2 proteins. Anti-LIN-14B antibody detects protein in extracts from wild- type animals but not in extracts from
lin-14(
n536n540) animals, which contain an amber mutation affecting all three predicted LIN-14 proteins, or from
lin-14(
n360) or
lin-14(
n355n534) animals, which were both expected to be molecular nulls for LIN-14B. Anti-C-terminal antibody did not detect altered size proteins in
lin-14(
n360) or
lin-14(
n355n534) extracts that would have suggested use of an alternative translational start site downstream of the mutant lesions. Anti-LIN- 14A antibody detects protein in extracts from all mutant back- grounds tested except for the LIN-14 nonsense mutant
lin-14(
n536n540). No truncated proteins were detected in lin- 14
(n536n540) extracts with any of the three anti-LIN-14 antibodies, suggesting that the altered transcripts were degraded by the nonsense-mediated decay mechanism of C. elegans.