Atf6 img 273

Samples were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA). For cytoplasmic fractions or whole cell lysates, 100 μg of total protein was loaded. For nuclear fractions, 10 μg of total protein was loaded. The following antibodies and conditions were used: cleaved PARP (9544; 1:500; Cell Signaling, Danvers, MA), caspase-6 (9762; 1:500; Cell Signaling), cleaved caspase-3 (9664; 1:500; Cell Signaling), XBP-1 (sc-7160; 1:200; Santa Cruz, Dallas, TX), ATF4 (sc-200; 1:200; Santa Cruz), ATF6 (IMG-273; 1:200; Imgenex, San Diego, CA); LRH-1 (PP-H2325-10; 1:200; Perseus Proteomics, Tokyo, Japan), pATF2 (9225S; 1:500; Cell Signaling [note: we have experienced difficulties with recent batches]), ATF2 (ab47476; 1:1000; Abcam, Cambridge, England); and PLK3 (4896S; 1:400; Cell Signaling). Immobilon Western substrate (Millipore, Billerica, MA) was used for detection.

Frozen liver fragments (50 mg) were homogenized in 500 μL of homogenization buffer: [7M Urea, 2M Thiourea, 4% CHAPs, 40 mM DTT, supplemented with protease and phosphatase inhibitors (Complete and Phostop, respectively; Roche, Mannheim, Germany)] and ultracentrifuged at 75.000 rpm for 45 min. Supernatants were collected in a clean cryo-tube and conserved in -80°C until use. Equivalent amounts of protein were separated in denaturing polyacrylamide gels and transferred onto a nitrocellulose membrane. The primary antibodies against p-eIF2α (Ser51-#9721), IRE1α (#3294), Irβ (#3025), AKT(#9272), p-AKT (Thr308-#9275) and p-AKT (Ser473-#9271) were purchased from Cell Signaling Technology, XBP1(sc7160) and EIF2α (sc11386) from Santa Cruz Biotechnology and ATF6 (IMG273) from IMGENEX, alpha-tubulin (T6074) from Sigma. Detection of immunolabeled proteins was performed using a commercial chemiluminescent assay (ECL prime; Amersham, Buckinghamshire, UK). Visualization and quantitative measurements were made with a CCD camera and software for Western blot image analysis (Odissey Fc Imager System and Image Studio Lite v 4.0, respectively; Li-COR, Bad Homburg, Germany).