Sc 1050

Sections (4 µm thick) were cut, air-dried, and deparaffinized through a series of xylene and graded ethanol (100%–70%). Heat-Induced Epitope Retrieval was performed in Retrieve-All-2 buffer (Signet Laboratories, Dedham, MA) pH 10, for 30 min followed by 0.1% Triton-X for 10 min at room temperature for CHOP staining [12] (link). No antigen retrieval was used for GRP78 staining [32] (link). Tissues were stained for CHOP to determine if treatment with TM or 4-PBA resulted in modifications in the expression of this transcription factor. Sections were incubated with either a rabbit anti-CHOP antibody (1∶40, sc-575; Santa Cruz Biotechnology) or a goat anti-GRP78 antibody (1∶40, sc-1050; Santa Cruz Biotechnology). The primary antibody was detected using a species-specific secondary antibody conjugated to an Alexa dye at 647 nm excitation wavelength, producing an emission maximum at 668 nm in the far-red region of the spectrum (1∶500; Invitrogen). This emission wavelength was used due to the high levels of autofluorescence in the renal tubular epithelium at the typical emission wavelengths for FITC (520 nm) or tetramethylrhodamine (580 nm). Sections were incubated with 100 ng/ml DAPI to label cell nuclei and mounted with Permafluor (Thermo Scientific).

The HSPA5 expressions in mRNA and protein were analyzed differentially in human normal and tumor tissues from the HPA database, which includes IHC-based expression for approximately 20 different types of common cancers in 216 cancer patients (maximum 12 patients in a group) 37 . The mRNA levels for HSPA5 in different normal tissues were obtained from the consensus datasets of three sources (HPA, GTEx and FANTOM5). Consensus normalized expression levels for 54 tissue types and 7 blood cell types were created from the above three datasets with the normalization pipeline (https://www.proteinatlas.org/about/assays+annotation#normalization_rna). Protein expression data were shown for each of the 44 normal tissues. Two antibodies for HSPA5 (cat #: CAB005221, sc-1050, Santa Cruz Biotechnology; or cat #: HPA038845, Sigma-Aldrich) were used for IHC staining in these data 40 (link).

C6/36 cells were grown in six-well plates and the cells were then incubated with 20 μg of a rabbit polyclonal anti-VDAC antibody (sc-98708; Santa Cruz Biotechnology Inc.) or 20 μg of a goat polyclonal anti-GRP78 antibody (sc-1050; Santa Cruz Biotechnology Inc.) or 20 μg of a combination of each antibody or 20 μg of a mouse monoclonal anti-αV/β5 integrin antibody (sc-13588; Santa Cruz Biotechnology Inc.) or no antibody at 28°C for 1. After incubation, the cells were incubated with JEV at MOI of 10 for 2 h at 28°C. The cells were washed with 1x  PBS and treated with acid glycine, pH 3, for 1 min to remove uninternalized virus [38 (link)]. The cells were washed with 1x  PBS and fresh growth medium was added. Cells were collected at 8 hours after infection and analyzed by flow cytometry as described previously [19 (link)]. Each sample was analyzed in duplicate and the experiment was done independently in triplicate.

Cells were grown in 10 cm dishes were resuspended in ice cold non-denaturing immunoprecipitation buffer containing 20 mM Tris HCL, 137 mM NaCl, 1% NP-40, 2 mM EDTA, and protease inhibitors (Roche). Total cell protein was normalized using a protein assay (BioRad) and 1 mg of protein from each sample was incubated with 2 µg of capture antibody targeted against GRP78 (Santa Cruz Biotechnology; SC-1050) and rotated on a platform for 24 h at 4 ˚C. Following incubation, samples were exposed to 100 µl of Protein G magnetic Surebeads (BioRad) for an additional 2 h on a rotating platform at 4 ˚C. Beads conjugated to the anti-GRP78 antibody were subsequently isolated and the remaining sample was collected and labeled “input” for use as controls. The magnetic beads underwent four consecutive washes using the non-denaturing IP buffer and resuspended and boiled in 100 µl of 4X SDS-PAGE sample buffer.

Coimmunoprecipitated proteins were separated by electrophoresis through 12% SDS-polyacrylamide gels and subsequently transferred to solid support (nitrocellulose membranes) using the Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories, Richmond, CA). The membranes were blocked with 5% skimmed milk in TBS at room temperature for 2 h. For western blot analysis, the membranes were incubated with a 1 : 1000 dilution of a goat polyclonal anti-GRP78 antibody (SC-1050; Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by a 1 : 2000 dilution of a HRP-conjugated rabbit anti-goat IgG antibody (31402, Pierce, Thermo Fisher Scientific Inc., Rockford, IL) at room temperature for 2 h. Final signal was detected by using the ECL Plus Western Blotting analysis kit (GE Healthcare).