Fluorescent dye labeled secondary antibody

The yeast whole-cell extract preparation was performed as previously described59 (link). The samples were collected and resuspended in 30 μl distilled water, and an equal volume of 0.2 M NaOH was added. Next, cells were collected after 10 min of incubation at room temperature, and the supernatant was carefully removed. Approximately 30 μl of SDS-sample buffer (100 mM Tris-HCL, pH6.8, 200 mM DTT, 4% SDS, 0.2% BPB, and 20% glycerol) was added to the pellet, and the cells were resuspended and boiled for 10 min and centrifuged briefly. The extract was loaded onto an SDS-PAGE gel and detected by immunoblotting with primary antibodies. The immunoblotting was performed using a fluorescent dye-labeled secondary antibody (Invitrogen), and the blots were scanned using an Odyssey infrared imager.

Epidermis and keratinocytes were lysed in denaturing RIPA lysis buffer supplemented with 20 mM N-ethylmaleimide (Sigma, USA), protease, and phosphatase inhibitors (Roche, Mannheim, Germany). SDS-PAGE (10%) was carried out using the mini-gel system from Bio-Rad. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in PBST buffer for 1 h at room temperature, then the membrane was incubated overnight at 4° C with specific primary antibodies against Fam114A1, RACK1, Bax, Bcl2, Caspase 3, cytc, P53, and GADPH/Actin (all 1:1000; Abcam, Cambridge, UK). The membrane was then washed with pBST buffer and incubated with fluorescent dye-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature in the dark. The proteins were visualized using an Odyssey Infrared Imaging System (LI-COR, Lincoln, Nebraska, USA).