Phospho ask1 thr845

Cells were rinsed with cold phosphate-buffered saline and homogenized in Laemmli SDS buffer. After separation and transferring to the PVDF membranes, proteins on the membranes were identified with the following antibodies: poly(ADP-ribose) polymerase 1 (PARP-1), extracellular signal-regulated kinase (ERK), phospho-ERK, c-Jun N-terminal kinase (JNK), phospho-JNK, p38, phospho-p38, Akt, phospho-Akt, PERK, phospho-PERK, eIF2α, phospho-eIF2α, CHOP, caspase 8, caspase 9, Bax, cytochrome oxidase IV (COX IV), Mcl-1, FLICE inhibiting protein (FLIP), apoptosis signal-regulating kinase 1 (Ask1), phospho-Ask1 Ser-83, phospho-Ask1 Thr-845 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (R&D Systems, Minneapolis, MN, USA), and β-tubulin (Sigma-Aldrich, St. Louis, MO, USA). The reaction products were determined with horseradish peroxidase-labeled IgG and visualized using enhanced chemiluminescence (ECL) Western blotting reagents. Finally, the signals were determined quantitatively with a computer image analysis system (IS1000; Alpha Innotech Corporation).

Small interfering RNAs (siRNAs) for NOX4, NOX1, Smad4, and ASK1 and antibodies against glyceraldehyde 3-phosphate dehydrogenase, phospho-HSP27 (Ser78), HSP27, Trx, GSTM1, AP1, SP1, Smad4, ASK1, and phospho-ASK1 (Thr845) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p38, phospho-p38 (Thr180/Tyr182), phospho-ERK (Thr202/Tyr204), phospho-src (Tyr416), phospho-Akt (Ser473), phospho-stat3 (Tyr705), phospho-p65 (Ser536), phospho-JNK (Thr183/Tyr185), phospho-Smad2 (Ser465/467), phospho-Smad3 (Ser423/425), Smad2, Smad3, p65, ERK, Akt, stat3, JNK, src, BIP, Calnexin, Ero1-Lα, CHOP, PERK, and PDI were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-IRE1α was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Peroxiredoxin (Prx) was purchased from Abfrontier (Seoul, Korea). Glutaredoxin (Grx) was purchased from Novus Biologicals (Littleton, CO, USA). Recombinant human TGF-β1 and TGF-β2 were purchased from R&D Systems (Minneapolis, MN, USA). SB203580 was purchased from EMD Millipore (Billerica, MA, USA), and GKT137831 was purchased from ApexBio (Houston, TX, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The total cellular samples were washed twice with PBS and lysed in RIPA buffer (HaiGene) supplemented with 1 mM PMSF (Sigma-Aldrich, St. Louis, MO). The concentration of total protein was determined using BCA Protein Assay Kit (Pierce, Rockford, IL). The total cellular protein extracts were separated by SDS–PAGE and transferred onto the nitrocellulose membrane (PALL, Washington, NY) in 20 mM Tris–HCl (pH 8.0) containing 150 mM glycine and 20% (v/v) methanol. The membrane was blocked with 5% nonfat dry milk in 1 × TBS containing 0.05% Tween 20 and incubated with primary antibody at 4 °C overnight. Antibodies against Akt (1:1000), phospho-Akt (Ser473) (1:500) were purchased from Cell Signaling Technology. Antibodies against ASK1 (1:500), phospho-ASK1 (Thr845) (1:500), p38 (1:1000), phospho-p38 (Tyr182) (1:500), β-Actin (1:1000) and secondary antibodies (1:5000) were all purchased from Santa Cruz (Weatherford, TX). Occludin antibody (1:1000) was obtained from Invitrogen, and ZO-1 antibody (1:1000) was obtained from Abcam (Cambridge, MA). The signals were developed with enhanced chemiluminescence reagent (HaiGene), and the digital images were captured using a LAS-4000 CCD camera system (Fuji film, Tokyo, Japan). The relative intensity of bands was analyzed by the software of Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD).