Grp78

Lysates from the hippocampus or primary culture were prepared using a lysis buffer (50 mM Tris, 150 mM NaCl, 0.02% NaN3, 0.5% Nonidet P-40, 0.5% Triton X-100, and protease inhibitor mixture), and protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Samples were subjected to 7.5% Tris-HCl, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot analysis, using antibodies against iNOS (BD Biosciences), COX-2 (BD Biosciences), GFAP (Millipore), GPR37 (Abnova), GRP78 (Novus), procaspase-3 (Stressgen), caspase-3 (Cell Signaling), caspase-12 (Stressgen), and CHOP (Abcam). An antibody against β-actin (Novus) was used to normalize the quantity of protein in each sample. Enhanced chemiluminescence (ECL; PerkinElmer) was used for the western blot detection. Corresponding bands were quantified with ImageJ (NIH imaging software), and one-way ANOVA was tested for significance. Data are presented as the mean ± SEM (the standard error of the mean). At least four litters from four pregnant rats of each group were used for all assessments.

CD34+ cells were grown in StemSpan SFEM II medium (Stemcell Technologies, Vancouver, BC, Canada) supplemented with StemSpan Myeloid Expansion supplement (SCF, TPO, G-CSF, GM-CSF) (Stemcell Technologies) and 1% penicillin/streptomycin in a humidified 5% CO₂ atmosphere at 37 °C. CD34+ cells were grown for 3 days in untreated medium followed by culture for 3 days in medium containing the human recombinant proteins GRP78 (#NBC-118378, Novus biologicals, Littleton, CO, USA), CALR (#NBP1-44499, Novus biologicals, Littleton, CO, USA), PDIA3 (#15922189, Thermo Fisher Scientific, Waltham, MA, USA) and GPI (#SAE0005, Merck KGaA, Darmstadt, Germany), respectively. All proteins were used at a final concentration of 500 pg/µL (equivalent to concentrations of [6.4–10.6 nM] according to molecular weight). CD34+ cells grown in control medium (for 6 days) not containing any of the recombinant proteins were used as control.