Goat anti mouse igg conjugated to horseradish peroxidase

The MSCs were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) to obtain total cellular protein. Cell lysates (20 µg of total protein) were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to the nitrocellulose membrane. After the blots had been washed with TBST (10 mM Tris-HCl (pH 7.60), 150 mM NaCl, 0.05% Tween-20), they were blocked with 5% skimmed milk for 1 h and incubated with appropriate primary antibodies at the dilutions recommended by the supplier. Antibodies against GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, IRE1α, CHOP, p-IRE1α, NF-κB, p-NF-κB, JNK, p-JNK, p38, p-p38, BCL-2, BAX, cleaved caspase-3, cleaved PARP-1, PrP^C, MnSOD, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The membranes were then washed, and primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).

To confirm the expression of PA-D4, 293T cells were plated at 1×10⁶ cells per well into six-well plates. Cells were transfected with 1 μg of plasmid DNA using the transfection agent Vivagen (Vivagen, Seoul, Korea) according to the manufacturer’s guidelines. After 48 h, cells were harvested and lysed. The cell lysates were separated by 4–20% polyacrylamide gel electrophoresis. Proteins were then transferred from the gel to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h in 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) and probed for 1.5 h with primary mouse anti-PA-D4 sera diluted 1:5,000 in TBST/5% nonfat skim milk. Membranes were then washed with TBST 3 times and incubated for 1 h with goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, USA) at a 1:5,000 dilution in TBST. After additional 4 washes with TBST, the bound antibody was visualized using Western Lighting Plus-ECL (PerkinElmer, Waltham, USA).
We also constructed plasmids that expressed PA-D4 antigens as fusion proteins with enhanced green fluorescent protein as described above. Expression of EGFP was determined using a confocal laser-scanning microscope (LSM 5; Carl Zeiss, Oberkochen, Germany) at 48 h after transfection.

Protein extracts were separated via 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membrane. After blocking membranes with 5% skim milk for 1 h, glucose-regulated protein 78 (GRP78) primary antibodies against PrP^C, protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), inositol-requiring enzyme 1α (IRE1α), p-IRE1α, activating transcription factor 4 (ATF4), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated overnight. After washing with PBS, the membranes were incubated with goat anti-rabbit immunoglobulin g (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).