Odyssey fluorescence imaging system

To investigate MUC1 expression on the surface of MUC1+B16 cell, MCF7 and MUC1+B16 cell surface protein were isolated using Cell Surface Protein Isolation Kit(Pierce) according to the standard manual. The isolated cell surface protein samples were analyzed by western blotting. Samples were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated overnight at 4°C with anti-MUC1 antibody(abcam, ab22711), followed by FITC-flourecent goat anti mouse IgG and finally visualized using Odyssey fluorescence imaging system(LICOR, US). To further investigate the binding profiles of anti-serum from the CTB-MUC1-alum-CpG group with native human MUC1, surface plasmon resonance analysis was conducted as previously described [40 (link)]. cells were blocked in phosphate-buffered saline (PBS) supplemented with 5% BSA and then incubated at 37°C with anti-serum from the CTB-MUC1-alum-CpG group. (1:100) for 1 h. The cells were then washed three times with PBS (pH 7.4) and then incubated at 37°C with PE-labeled rat anti-mouse IgG1 for another 1 h and fixed before labeling the nuclei with Hoechst 33258. Next, the cells were observed with a flouresent microscope (Zeiss).

The nuclear proteins and total proteins were isolated as previously described [16 (link)]. The protein levels of target genes were detected using specific primary antibodies and IRDye secondary antibodies on an Odyssey Fluorescence Imaging System (LI-COR Biosciences, USA) as described previously [19 (link), 35 (link), 36 (link)]. The density of the immunoblots was analyzed using Odyssey V3.0 software

Cell lysates were mixed with Laemmli buffer plus dithiothreitol (DTT), heated for 5 min at 95 °C, and loaded onto SDS-PAGE (sodium dodecyl sulfate—polyacrylamide gel electrophoresis) gels. The gels were run in Tris–Glycine SDS running buffer and then transferred onto nitrocellulose membranes. Blocking consisted of at 5% skim milk or 2.5% BSA, depending on the primary antibody used. Imaging was performed using the Li-Cor Odyssey fluorescence imaging system. Primary antibodies consisted of: monoclonal mouse anti-6xHis-Tag (Invitrogen, MA1-21315; 1:4000), polyclonal rabbit anti-ATF6 (Novus Biologicals, NBP1-75478; 1:1000), monoclonal rabbit anti-BiP (Cell Signaling Technology, C50B12; 1:1000), monoclonal rabbit anti-Calnexin (Cell Signaling Technology, C5C9; 1:1000), monoclonal rabbit anti-COG5 (Sigma-Aldrich, SAB4200440; 1:600), monoclonal mouse anti-GAPDH (Invitrogen, ZG003; 1:4000), monoclonal mouse anti-GFP (Cell Signaling Technology, 2955S; 1:1000), polyclonal rabbit anti-IRE1a (Cell Signaling Technology, 3294S; 1:1000), monoclonal rabbit anti-PDI (Cell Signaling Technology, 2446S; 1:1000), and monoclonal rabbit anti-PERK (Cell Signaling Technology, D11A8; 1:1000). Secondary antibodies consisted of: Donkey anti-Rabbit IRDye 680, Donkey anti-Rabbit IRDye 800, Donkey anti-Mouse IRDye 680, and Donkey anti-Mouse IRDye 800 (Li-Cor; 1:20,000).

Cultured cells were lysed using RIPA lysis buffer supplemented with phosphatase and protease inhibitors (Roche Diagnostics, Basel, Switzerland). Total protein was quantified using a bicinchoninic acid assay (Thermo Fisher Scientific) and then equal quantities of extracted protein (20–30 µg) were separated via 12 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto 0.22-µm PVDF membranes (MilliporeSigma, Burlington, MA, USA). The membranes were blocked using 5% BSA-PBS (Beyotime Biotechnology) at room temperature (RT = 25 °C) for 1 h, and then incubated with primary antibodies (Cleaved caspase-3, Cleaved PARP, BAX, BCL-2, Collagen II, MMP13, ADAMTS5). The membranes were washed with Tris-buffered saline (TBS)-0.1% Tween 20 (TBST) and subsequently incubated with anti-rabbit IgG (H + L) secondary antibody (cat. no. 5151; DyLight™ 800 4X PEG Conjugate; Cell Signaling Technology; 1:5000) for 1 h at RT in the dark. After washing in TBST, protein immunoreactivity was detected using the Odyssey Fluorescence Imaging system (LI-COR Biosciences, Lincoln, NE, USA). Semi-quantitative analysis of protein band intensity was conducted using the ImageJ v1.8.0 software (National Institutes of Health) and normalized to the intensity of the internal loading control, β-actin.

To determine the potency and selectivity of the Cy5-probes, each active site-titrated cathepsin was preincubated separately in the assay buffer for 15 min at 37 °C at a concentration of 10 nM (experiment A) or 100 nM (experiment B), and then mixed with various concentrations of either the MP-cL3 or MP-pc1 probe for 30 min. In experiment A, the probe concentrations were 2 nM, 10 nM, 50 nM and 250 nM, while in experiment B the probe concentrations were 20 nM, 100 nM, 500 nM and 2.5 μM. The reaction volume for all the samples was 200 μL. After 30 min, 100 μL of 3× SDS/DTT was added to the reaction mixtures, the samples were boiled for 5 min, and then 20 μL of the sample was run on 4–12% Bis-Tris Plus 15-well gels for 30 min at 200 V with 2 μL of PageRuler™ Prestained Protein Ladder. The gels were then directly scanned at 700 nm (red channel for Cy5 detection; excitation 685 nm) using an Odyssey fluorescence imaging system (LI-COR) and the images were analyzed using Image Studio software.