Synergy 2 machine

Harvested cells from each experiment were suspended in RIPA buffer with a proteinase inhibitor cocktail (Roche Complete Tablet) and sonicated until cell membranes were fully disrupted to release protein. The protein was then quantified using BSA (bovine serum albumin) standards on a Biotek Synergy 2 machine. After quantification, samples were mixed with NuPAGE LDS Sample Buffer 4X (Invitrogen) to make a 1X concentration with 5% beta-mercaptoethanol. Protein was loaded in an SDS-PAGE gel with percentages adjusted to detect the target protein of interest. Precision Plus Protein Kaleidoscope (Bio-Rad) was used as a ladder for all immunoblots. After running the gel, protein was transferred onto a nitrocellulose membrane overnight using a wet transfer buffer at 30 V. The membrane was blocked for 30 min in 5% skim milk in TBS at room temperature. Primary antibodies were diluted either in 5% BSA or 5% skim milk in TBS and incubated overnight at 4°C, or 5 h at room temperature. Membranes were washed 3 times in TBS-T. Secondary antibodies (anti-mouse or anti-rabbit) were diluted in 5% skim milk in TBS and membranes were incubated for 1 h at room temperature in the absence of light. Membranes were washed again in TBS-T, and lastly imaged on a LI-COR imager for analysis.

Cytotoxicity assay was detected using Cell Counting Kit-8 (Dojindo, Japan). 10 μL of CCK-8 solutions was added to 100 μL cell culture medium. After incubation at 37°C for 4 h, the absorbance at 450 nm was measured with Synergy2 machine (BioTek).