Figure 2. Phosphorylation of PERK during brown adipocyte differentiation.

(A, B, C) Phosphorylation of PERK in differentiating cells. (A, B) Cells were cultured with differentiation medium (A, − [control]) or differentiation enhancement medium (A, + and B) for the indicated number of hours on day 2. (A) Cells treated with (+) or without (−) 40 nM thapsigargin (Tg) for 1 or 6 h on day 2 were included as positive controls (A). (A, B) The cell lysates were analysed by immunoblotting (IB) with the indicated antibodies (A, B). (C) Lysates from cells cultured with differentiation enhancement medium for 12 h on day 2 were incubated with or without 2 units of λ phosphatase (PPase) at 30°C for 30 min and analysed by IB with the indicated antibodies (C). (B) The expression of GRP78 was calculated and is shown as the ratio relative to actin expression (B). (D) Newly synthesized proteins in differentiating cells. Cells were incubated with 10 μg/ml puromycin for 10 min and lysed at the indicated time points on day 2. Newly synthesized proteins were detected by IB with an anti-puromycin antibody. (E) IB analysis of ATF4 in differentiating cells. Cells were harvested after the indicated number of hours on day 2 and analysed by IB using the indicated antibodies. Cells treated with 40 nM Tg for 6 h on day 2 were included as positive controls. (F, G) Expression of CHOP (F) or Gadd34 (G) mRNA in differentiating cells. Cells were harvested after the indicated number of hours on day 2, and total RNA was isolated. The data are shown as the fold change relative to the value at 0 h (n = 3 independent experiments). Cells treated with 40 nM Tg for 12 h on day 2 were included as positive controls brown adipocytes.