Ecl substrate

Total protein was collected and lysed applying RIPA buffer (ProMab Biotechnologies, Inc., Richmond, CA, USA) based on the manufacturer’s instructions. The proteins were separated via 10% SDS–PAGE and then transferred to polyvinylidene fluoride membranes. Next, the PVDF were incubated with primary antibodies (GAPDH, 1:2000, ab8245; CPEB3, 1:500, ab10883) at 4°C overnight, followed by incubation with the second antibody at room temperature for 1.5 h. Eventually, the signals were visualized via ChemiScope 3300 Mini (Clinx) applying the ECL substrate (Cyanagen).

HEK293T cells were transfected with the indicated plasmids and after 2 days, lysates were prepared [lysis buffer contained phosphate-buffered saline (PBS), 10 mM Na⁺-pyrophosphate, 50 mM NaF, and pH was adjusted to 7.4. 1 mM Na⁺-orthovanadate, 1% Triton X-100, and a cocktail of protease inhibitors (Roche) were freshly added before each use]. The cells were placed on ice and scraped after addition of lysis buffer. Protein extracts were incubated with Protein G Sepharose beads (Sigma-Aldrich, St. Louis, MO) and anti-His antibody (1:100) (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C. The beads-protein complexes were incubated for 4 h at 4°C, centrifuged, and washed with lysis buffer four times. Samples were prepared for running on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by heating (37°C for 30 min) in SDS sample buffer. Subsequently, the samples were transferred to nitrocellulose membranes and incubated overnight at 4°C with anti-SLC26A6 (1:500) (ab 172684, Abcam) and the next day exposed to the appropriate secondary antibody. Signal was developed using enhanced-chemiluminescence (ECL) substrate (CYANAGEN).