(A) K562 cells were cultured in the presence of DMSO, hemin, or PMA for 72 hours, as described in Methods. Cells lysates were subjected to Western blot analysis with antibody against tescalcin. (B) Stimulation with PMA upregulates tescalcin at the mRNA level. K562 and HEL cells were cultured in the absence or presence of PMA for 72 hours. Total RNA subjected to quantitative RQ-PCR with tescalcin-specific primers (TaqMan). Obtained values (n = 3; mean ± SD) were normalized to 18S ribosomal RNA, and expressed relative to unstimulated K562. (C) Tescalcin expression in primary MKs. Mature mouse MKs were obtained from fetal livers as described in Methods. Lysates of fetal liver cells (FLC) and MKs were analyzed for tescalcin expression by Western blot. K562 lysate was used as positive control. (D) K562 cells were cultured in the presence of PMA (10 nM) for the indicated times, and the accumulation of tescalcin was determined by Western blot. (E) Bryostatin blocks PMA-induced upregulation of tescalcin. K562 cells were stimulated by 10 nM PMA in the absence or presence of 100 nM bryostatin (Bryo). The expression of tescalcin was determined by Western blot. (F) Sustained ERK activity is required for tescalcin upregulation. K562 cells were stimulated by PMA (10 nM) in the absence or presence of MEK1/2 inhibitor (20 μM; U0126). Cell lysates were probed with antibodies to tescalcin, total p44/42 MAPKs (ERK1/2), and phospho-p44/42 MAP kinases (ERK1*/2*).