Irdye odyssey secondary antibodies

To confirm the expression of FJX1 after transfection, transfected HeLa/T cells were lysed on ice in RIPA buffer (50 mM Tris pH8, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, 150 mM NaCl) containing the protease inhibitor cocktail (Roche, USA). Cell lysates were collected after centrifugation at 14000 rpm for 15 minutes at 4°C, and the concentration of the total cellular protein for each sample was determined using the Bradford protein assay (Pierce Biotechnology, USA). Extracted proteins (50 μg) were denatured at 95°C for 5 minutes followed by a 12% SDS-polyacrylamide gel separation, and separated proteins were transferred onto the Immobilon-P membrane (Millipore, MA, USA). Then, blots were blocked with 5% skimmed milk in TBST for 1 hour and further incubated with the indicated primary antibodies (anti-V5: 1 : 2000; Abcam, UK; anti-α-tubulin: 1: 2000; Sigma-Aldrich, USA) overnight at 4°C. The next day, blots were washed (3 times for 5 minutes each) in TBST buffer and subsequently incubated with the relevant IRDye Odyssey secondary antibodies (LI-COR Biosciences, USA) for 1 hour. After washing (TBST), detection was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences, USA). Due to the unavailability of a good antibody against FJX1, an anti-V5 antibody was used to detect FJX1 overexpression after transfection.