Fig 1: Cel protein is differentially distributed among subcellular fractions and total pancreas tissue from ethanol-fed Xbp1+/- or WT mice, and in cultured AR42J pancreatic acinar cells exposed to ethanol. Pancreas tissue was harvested and fractionated as in Figure 1, from WT or Xbp1+/- mice fed control (con) or ethanol (EtOH) diets for 5–6 weeks. Fractions were obtained by centrifugation at (1) 1500 × g; (2) 10,000 × g; and (3) 85,000 × g. (A) Cel (70 kilodalton) protein was analyzed by Western blot (upper panels), and quantitated in the different fractions (20 μg/lane) in the graph (lower panels) (optical density, means ± SEM, n = 3). A Cel protein fragment of ∼48 kilodaltons also was detected in the fractions. (B) Cel protein expression is enhanced in total pancreas tissue upon EtOH feeding or in the Xbp1+/- mice. Pancreas tissue was harvested from control- or EtOH-fed WT or Xbp1+/- mice, and total proteins extracted with RIPA buffer without further fractionation. Cel protein and extracellular signal-regulated kinase 1/2 as a loading control were analyzed by Western blot in tissue extract (40 μg/lane). In the graph, the optical density of Cel bands was quantitated relative to total extracellular signal-regulated kinase protein; data indicate means ± SEM, n = 3. (C) AR42J cells were cultured for 48 hours in control medium (K12 medium supplemented with 12% fetal bovine serum) alone or with 100 mmol/L EtOH. ER membrane fractions were obtained by centrifugation of hypotonically lysed cells at (1) 150 × g; (2) 750 × g; (3) 1300 × g; (4) 5000 × g; (5) 12,000 × g; and (6) 100,000 × g. Cel protein in the fractions (10 mg/lane) was analyzed by Western blot. Arrows indicate the following: (A) Rat Cel, ∼70 kilodaltons; and (B) Cel fragments, ∼40–60 kilodaltons. (D) Ethanol-induced aggregate formation in AR42J cells. Cells were kept untreated or treated with 100 mmol/L EtOH for 48 hours, and then cellular aggresomes were visualized in a confocal microscope using the PROTEOSTAT Aggresome detection kit (ENZ-51035; Enzo Life Sciences, Farmingdale, NY). Red fluorescence (left panels) indicates aggresomes; corresponding differential interference contrast microscopy images are shown (right panels). ∗, P value less than .05 by student's t test.