Figure S4. Phosphorylation sites of PERK during brown adipocyte differentiation.

(A) Immunoblotting (IB) analysis of exogenously expressed PERK-ΔLD in differentiating cells. siPERK-transfected cells were infected with retroviruses expressing PERK-ΔLD. The cells were lysed after the stimulation with 40 nM Tg or 2.5 μg/ml tunicamycin (Tm) for 1 h on day 2 and analysed by IB with the indicated antibodies. Cells treated with differentiation enhancement medium for 12 h on day 2 were included as positive controls. (B) LC-MS/MS–based phosphoproteomic analysis of phosphorylation sites of PERK-ΔLD-KA during brown adipocyte differentiation. The LC-MS/MS data revealed a peptide (⁷¹⁴RSRSFSVGISCGQTSSSE⁷³¹) that included phospho-Ser719. Ser719 is highlighted in red. (C) IB analysis of the phosphorylation of endogenous PERK during ER stress in differentiating cells. Cells were stimulated with or without 40 nM Tg or 2.5 μg/ml Tm for 1 h on day 2. The cell lysates were analysed by IB with the indicated antibodies. Cells treated with differentiation enhancement medium for 12 h on day 2 were included as positive controls. An arrowhead indicates the phosphorylated PERK at Ser715, Ser717, and/or Ser719.