Grp94

Total protein content of the samples was assessed by a microplate protein assay (Bio-Rad, Hercules, CA), and equal amounts of protein per sample and known molecular weight markers were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto PVDF membranes (Whatman, Florham Park, NJ) and incubated for 1 h at room temperature with a blocking solution (3% bovine serum albumin) in Tris-buffered saline buffer containing 0.1% Tween 20 (TBST). The blocked membranes were incubated with primary antibodies for ERK, phospho-ERK, IRE1α and PDI (Cell Signaling), GRP94 (Enzo Life Science), GRP78 (BD Japan), phospho-eIF2α (Ser52) (Invitrogen), phospho-IRE1α (Ser724) (Novus Biological) and β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and washed three times with TBST. The membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (GE Healthcare, UK) for 1 h at room temperature and washed. Immunodetection analyses were performed using a BM Chemiluminescence Blotting Substrate (POD) Kit (Roche Diagnostics, Mannheim, Germany).

Cell lysates were prepared as described previously and precleared with protein A/Sepharose beads (Merck‐Millipore) for 1 h at 4 °C after which the protein concentration was measured as described previously. Precleared lysate (0.5 mg) was added to 50 μL of antibody‐bead slurry and rotated overnight at 4 °C. Antibody complexes were washed thrice with lysis buffer and then resuspended in sample buffer. acetylated lysine residues of a number of HSP90 isoforms were determined by SDS/PAGE and western blotting. Antibodies used included HSP90 (ADI‐SPA‐835), GRP94 (ADI‐SPA‐850) and TRAP1 (ADI‐SPA‐971) obtained from Enzo Life Science, and acetylated lysine (#9441) was obtained from Cell Signaling Technologies.

Immunostaining on paraffin-embedded or frozen tissue sections was performed as described previously [13 (link), 42 (link)]. Tissue sections were incubated at 4°C overnight with primary antibodies against GRP94 (1:250, Enzo Life Sciences, Farmingdale, NY), cytokeratin 14 (1:400), p63 (1:200), Cyclin D1 (1:100), Ki67 (1:200) from Thermo Scientific (Fremont, CA), β-catenin (1:100, Santa Cruz Biotechnology, Dallas, TX), active β-catenin (1:50, Millipore, Billerica, MA), phospho-S6 (1:200), S6 (1:50), phospho-AKT (Ser 473, 1:50), AKT (1:200), phospho-Histone H3 (1:200) from Cell Signaling Technology (Danver, MA), cytokeratin 8 (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA), α-Smooth Muscle Actin (1:2000, Sigma-Aldrich, St. Louis, MO), pan cytokeratin (1:50, Abcam, Cambridge, MA) and E-cadherin (1:50, BD Biosciences, San Jose, CA). Immunofluorescence was analyzed using a Zeiss LSM 510 confocal microscope with LSM 510 Version 4.2 SP1 acquisition software. Confocal images were acquired with 20X or 40X oil lens. Images were then processed with LSM Image Browser R4.2 and Adobe Photoshop CS5.
Staining for mucin by either mucicarmine or periodic acid-Schiff stain (PAS) followed by diastase digestion was performed by the clinical histology laboratory at University of Southern California using standard protocols.

Tissue lysates were subjected to SDS-PAGE and Western blot analysis as described previously [41 (link)]. Primary antibodies used were GRP94 (1:5000, Enzo Life Sciences, Farmingdale, NY), PTEN (1:1000, Cell Signaling, Danver, MA) and GAPDH (1:5000, Santa Cruz Biotechnology, Dallas, TX).