SDS-PAGE and western blot analysis

For SDS-PAGE and western blot analysis, collect cells by trypsinization and subsequent centrifugation at 300x g,4°C.

Wash the cell pellets in PBS and centrifuge once more at 300x g,4°C.

Remove the supernatant and lyse the cell pellets in RIPA buffer supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP, 4906837001, Roche).

After incubating in RIPA buffer for 0h 20m 0s On ice, clear the samples by centrifugation at 20000x g,4°C.

Collect the soluble supernatant fraction and measure the protein concentrations using the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).

Then, adjust the samples for equal loading and mix it with 6x protein loading dye, supplemented with 100 mM DTT. Then, boil it for 0h 5m 0s at 95°C.

Load the samples on 4-12% SDS-PAGE gels (NP0321BOX, NP0322BOX, or NP0323BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).

Transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for 1h 0m 0s at 4°C using the Mini Trans-Blot Cell (Bio-Rad).

After the transfer, block the membranes with 5% milk powder dissolved in PBS-Tween (0.1% Tween 20) for 1h 0m 0s at Room temperature.

Incubate the membranes 1h 0m 0s at 4°C with primary antibodies dissolved in the blocking buffer and wash.

Wash for 0h 5m 0s.(1/3)

Wash for 0h 5m 0s.(2/3)

Wash for 0h 5m 0s.(3/3)

Then, incubate with species-matched secondary horseradish peroxidase (HRP)-coupled antibodies diluted 1:10,000 in blocking buffer for 1h 0m 0s at Room temperature.

Afterwards wash the membranes three times with PBS-T and further process for western blot detection.

Incubate the membranes with SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) and image it with a ChemiDoc MP system (Bio-Rad).

Analyze the images with ImageJ [57].