Protein reagents

Cells were detached with Trypsin (0.25%) at 37 °C, washed with Phosphate Buffer Saline (PBS), harvested, and stored at −80 °C until processed. Cells were lysed with ice-cold lysis buffer and incubated on ice for 30 min. Whole cell lysates were centrifuged, supernatants were collected, and protein concentration was determined using Bio-Rad Protein Reagents (Bio-Rad, Hercules, CA, USA). In all cases, protein lysates (50 μg) were separated by SDS-PAGE (12% Acrylamide), blotted onto nitrocellulose membranes, and probed with the appropriate dilution (1:1000) of primary antibody (Sigma, St. Louis, MO, USA; Cat number AV3476). The membranes were rinsed and then incubated with mouse or rabbit IgG horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling, 1:5000 dilution). Bound antibodies were detected using enhanced chemiluminescence (GE Healthcare, Logan, UT, USA) followed by autoradiography in a FluorChem^TM 8900 (Alpha Innotech Corporation, San Leandro, CA, USA). The intensity of each band was quantified and recorded by Image Lab software (BioRad, Hercules, CA, USA).

Cells were collected, washed twice with Phosphate Buffer Saline (PBS), and stored at −80°C until processed. For total protein extraction, cells were lysed with a lysis buffer (150 mM NaCl, 1% Triton X-100, 0.4 mM NaF, 0.4 mM NaVO₄, 25 mM Tris-HCl, pH 7.6 and 1× protease inhibitor) for 45 min on ice and then centrifuged for 10 min at 4°C. The supernatants were collected for further analysis. Protein concentration was determined using Bio-Rad Protein Reagents (Bio-Rad, Hercules, CA). Protein lysates (50 μg) were separated by SDS-PAGE, blotted onto membranes, and probed with the appropriate dilution of each primary antibody. Membranes were rinsed and incubated with a horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using enhanced chemiluminescence (ECL) regents (GE Healthcare, Piscataway, NJ) and autoradiography using a FluorChem^™ 8900 (Alpha Innotech Corporation, San Leandro, CA). The primary antibodies used were: anti-RBPMS (24 kDa), anti-RCBTB1 (58 kDa), anti-ZNF608 (162 kDa) (Novus Biologicals, Littleton, CO), and anti-β-actin (42 kDa) (Sigma, St. Louis, MO). The secondary antibodies used were anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP) (Cell Signaling, Beverly, MA).

Following transient transfection of OMIs, the cells were detached with trypsin (0.25%) at 37 °C, washed with phosphate-buffered saline (PBS), harvested, and stored at −80 °C until processing. Cells were lysed with ice-cold lysis buffer and incubated on ice for 30 min. Whole-cell lysates were centrifuged, supernatants were collected, and protein concentrations were determined using Bio-Rad Protein Reagents (Bio-Rad). In all cases, protein lysates (30–50 µg) were separated by SDS-PAGE (12% acrylamide), blotted onto nitrocellulose membranes, and probed with appropriate dilutions of primary antibodies against PTEN (Cell Signaling, Danvers, MA; 9569P), ZEB1 (Novus, Centennial, CO; 23484SS), FOXO1 (Cell Signaling 2880S), and SNAI2 (Cell Signaling 9585S). β-Actin was used as a loading control (Sigma Aldrich A5441). The membranes were rinsed and incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cell Signaling). Bound antibodies were detected using enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) followed by autoradiography in a ChemiDoc Imaging System (Bio-Rad).