Pierce ecl chemiluminescence system

The treated cOTSC were washed with pre-warmed 1xPBS (pH 7.4) and sonicated in ice-cold lysis buffer (in mM: 50 Tris–HCl (pH 7.5), 150 KCl, 1 DTT, 1 PMFS, 0.1 % NP40, 1 % SDS, and complete protease inhibitor cocktail (Roche, #1187380001)). The lysates (40 μg) were subjected to gradient SDS-PAGE under reducing conditions (15–4 % Tris TGX gels; Bio-Rad, #456-1086), transferred onto PVDF membrane (0.2 μm pore size; Bio-Rad, #162-0177) and incubated overnight (4 °C) with anti-calbindin D_28K, anti-calpain-1, anti-calpain-2, anti-Cav2.1, anti-PKCγ, and anti-β-tubulin. The blots were developed using horseradish peroxidase-conjugated 2nd Ab (polyclonal Swine Anti-Rabbit IgG-HRP, Dako, #P0217; Goat Anti-Mouse IgG-HRP, Santa Cruz Biotechnology Inc., #SC-2500; 90 min, 22 °C) and the Pierce ECL chemiluminescence system (Thermo Fisher Scientific Inc., #3210634075). Semi-quantitative densitometry analysis was performed (Bio-Rad, Quantity One 4.6.6). For each sample, the protein level was normalized to β-tubulin and expressed relative to untreated naive control, set at 1 (arbitrary units).

From each cell pellet lysate, plasma membrane, MV, and EX fraction, a total of 3 µg of protein were subjected to 4–20% mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) and transferred to a PVFD membrane (Bio-Rad). After 1 h of blocking in 5% nonfat milk in PBS containing 0.1% Tween-20 (PBS-T) at room temperature, membranes were incubated overnight with antibodies. Membranes were washed in PBS-T and incubated with species-specific horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After a second washing with PBS-T, a Pierce ECL chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA) was used to develop membranes to detect bound antibodies. GAPDH served as the internal control for the CP and PM fractions, while ALIX and HSP70 served as internal controls for MVs and EXs which do not express GAPDH.

Cells were harvested and lysed on ice for 10 min in RIPA buffer (50 mm Tris, 150 mm NaCl, 0.5% EDTA, 0.5% NP‐40) and centrifuged for 15 min at 13 000 g. The concentration of total proteins was quantified by a colorimetric assay. 30 μg of total proteins was loaded and separated on 6%, 8%, or 10% SDS/PAGE gel and then transferred to poly(vinylidene difluoride) membrane. After blocking with 5% nonfat milk for 2 h at room temperature, the membrane was incubated with primary antibodies overnight at 4 °C. The antibodies used were antibodies to Plac1 (1 : 1000; Abcam), Furin (1 : 1000; Abcam), NICD (1 : 1000; Abcam), HES1 (1 : 1000; Cell Signaling Technology), PTEN (1 : 1000; Cell Signaling Technology), p‐AKT (1 : 1000; Cell Signaling Technology), MMP2 (1 : 1000; Abcam), and MMP9 (1 : 1000; Abcam). The membranes were incubated with secondary antibodies for 2 h, and proteins were then detected using the Pierce ECL chemiluminescence system (Thermo Fisher Scientific). The total proteins were extracted from MCF‐7 and MDA‐MB‐231 cells for co‐immunoprecipitation using IP/CO‐IP Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The bound proteins were analyzed by western blotting and proteome analysis.