FIGURE 6:

TIS11b is phosphorylated at S54 and S334 in hormone- and hypoxia-stimulated cells. (A) BAC cells were stimulated with ACTH in the presence or absence of H89 for the indicated periods of time. Total-cell extracts were probed for total TIS11b/TIS11d, pS54-TIS11b, and pS334-TIS11b. Owing to the conserved PKA consensus motifs (RRHS and RHS; see Figure 2) within TIS11b and TIS11d sequences, both phosphoproteins were detected. Note the shift of TIS11b toward high-molecular-weight species. The asterisk indicates the phospho-(S334)-TIS11b species appearing at 6 h poststimulation by ACTH. (B) A549 lung carcinoma cells were exposed to normoxia (0 h) or hypoxia for 2, 4, and 8 h. Cells extracts were probed by Western blot as described in A. (C) A549 cells were exposed to normoxia or hypoxia for 8 h in the absence or presence of OA, an inhibitor of the phosphatases PP2A/PP1. Note the pS334-TIS11b species appearing under hypoxia in the presence of OA (indicated by an asterisk). Blots were subsequently probed with an anti–β-actin to assess equal loading of samples.