Horseradish peroxidase hrp tagged secondary antibodies

Total cell lysates prepared from treated and untreated cells were harvested at defined time-points and subjected to Western blotting analysis as previously described [30 (link),32 (link),41 (link)]. Primary antibodies were purchased from commercial vendors and used for detecting human HuR, p27, BCL-2, alpha-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), cyclin D1, cyclin E1, HIF1-α, MITF, VEGF-A, Caspase-9, and PARP (Cell Signaling Technology Inc., Beverly, MA, USA) and beta-actin (Sigma Aldrich, St. Louis, MO, USA). Protein bands were detected using appropriate horseradish peroxidase (HRP)-tagged secondary antibodies (Santa Cruz Biotechnology) and an enhanced chemiluminescence kit (Thermo Scientific, MA, USA). Protein expression levels were detected on a chemiluminescence imaging system (Syngene, Frederick, MD, USA), and the relative protein expression compared to beta-actin or alpha-tubulin was quantified using Gene Tools software (Syngene) as previously described [30 (link),32 (link),41 (link)]. Experiments were repeated at least three separate times for reproducibility, and the data were analyzed for statistical significance.

Total protein extracted from cells that underwent various treatments was subjected to western blot analysis as previously described [15 , 37 , 56 ]. Briefly, protein samples were separated in 10 % SDS polyacrylamide gel and were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobiloin®, Millipore, MA, USA). After the transfer, the membranes were blocked in 5 % milk containing buffer (1X Tris buffered saline with Tween 20^® [TBST]) for 30 min. In the following step, membranes were incubated with primary antibodies against human HuR, p27, cyclin D1, cyclin E, Bcl-2 (Santa Cruz Biotechnology, Dallas TX, USA), Folic acid receptor-alpha (GeneTex Inc., Irvine, CA, USA), caspase-9 and PARP (Cell Signaling Technology Inc., Beverly, MA, USA), and beta-actin (Sigma-Aldrich, St. Louis, MO, USA), respectively, in 5 % milk containing TBST. Protein bands were detected using appropriate horseradish peroxidase-(HRP)-tagged secondary antibodies (Santa Cruz Biotechnology) and an enhanced chemiluminescence kit (Thermo Scientific, MA, USA). Protein expression levels were detected using a chemiluminescence imaging system (Syngene, Frederick, MD, USA) and quantified using Gene tools (Syngene) software [37 ].