Secondary anti mouse antibody

Fibroblasts were lysed in RIPA buffer (ThermoFisher) containing protease inhibitors (Roche) and sonicated for 5 minutes with the Bioruptor sonication device (Diagenode). Cell lysates were centrifuged at 13,000 x g for 10 minutes at 4 °C, and the supernatant was collected for protein quantification (Pierce BCA Protein Assay Kit). 30 μg of protein sample was mixed with Bolt™ LDS Sample Buffer and Sample Reducing Agent (ThermoFisfer) and heated at 90°C for 5 min. Samples were loaded on Bolt® 4–12% Bis-Tris Plus mini-gel followed by transfer into a nitrocellulose membrane (Bio-Rad). Membrane was blocked with 5% non-fat milk and incubated with anti-SORD (1:1000 dilution, ab189248, Abcam) antibody for 2 hours, washed with TBS containing 0.01% Tween 20 (Bio-Rad) and incubated with a peroxidase-conjugated anti-rabbit antibody (1:2500 dilution, Cat. #7074, Cell Signaling). Membrane was subsequently incubated with monoclonal antibody against tubulin (1:1000 dilution, Santa Cruz) and secondary anti-mouse antibody (1:2500 dilution, Cat. #7076, Cell Signaling). Chemoluminescence detection was performed with the SuperSignal™ West Pico PLUS Chemiluminescent Substrate and imaged with the FluorChem E (ProteinSimple).

Cell lysates were harvested from a panel of breast cancer cell lines (MCF7, T47D, MDA-MB-468, BT549, MDA-MB-231, and HCC1954). Proteins (15 μg) were separated using NuPAGE 4–12% Bis-Tris mini gels (Invitrogen). The gel was transferred to polyvinylidene difluoride (PVDF) membranes, probed with CD40 (R&D), NE (Santa Cruz), and actin (Cell Signaling) antibodies, and detected with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). The secondary anti-mouse antibody was obtained from Cell Signaling Technology (Danvers, MA).