Revert 700 stain

Cells were harvested with RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors (Roche). Total protein was separated on a 4–20% gradient SDS gel (Bio‐rad) and was transferred to a nitrocellulose membrane(Bio‐rad). The membranes were blocked with 5% non‐fat milk in Tris‐buffered saline solution and probed with primary antibodies overnight at 4°C. To ensure equal loading and transfer, membrane total protein staining was performed using Revert™700 stain (Li‐COR Biosciences) following the manufacturer's instructions. Immunoblot image acquisition and analysis were performed with Image Studio software (Li‐COR Biosciences).

Whole cell lysates were prepared in RIPA buffer supplemented with phosphatase and protease inhibitors (10 mM NaF + 1 mM NaOV + 1X PIC + 10 mM Na.pyrophosphate + AEBSF + 10 mM β-glycerophosphate). Cells were incubated in the RIPA solution on ice for > 20 min. Lysates were centrifuged at 14,000 rpm to remove cellular and nuclear debris. Bradford assay was used to determine protein concentration, and samples were heat denatured for 5 min at 95 °C in 4X loading buffer.
SDS-PAGE was used to resolve equal amounts of proteins, and the proteins were transferred to nitrocellulose. Revert 700 Total Protein stain (LI-COR Biosciences, UK) was used to reversibly stain the nitrocellulose membranes. Membranes were scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences, UK). After washing off the Revert, membranes were blocked with Odyssey Blocking Buffer TBS at room temperature for approximately one hour. Primary antibodies were diluted in Odyssey Blocking Buffer TBS, and membranes were incubated with the primary antibody at 4 °C overnight. IRDye-conjugated secondary antibodies were used for detection with the Odyssey system. Secondary antibody incubation was carried out for one hour. LI-COR Image Studio software was used for densitometry, and bands were normalised against the Revert 700 stain for that sample, according to the manufacturer’s instructions.