Rabbit anti eif4g antibody

Yap protein levels were determined by western immunoblotting analysis. In brief, proteins isolated from cells (40 μg) were separated by electrophoresis on a 10% SDS polyacrylamide gel. Partitioned proteins were transferred to a nitrocellulose membrane. The membrane was probed with rabbit anti-Yap antibody (Cell Signaling Technology), rabbit anti-PABP antibody (Abcam), rabbit anti-eIF4G antibody (Cell Signaling Technology), mouse anti-eIF4A, anti-eIF4B or anti-eIF4E (Santa Cruz Biotechnology). HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson ImmunoResearch) was used as the secondary antibody. The corresponding protein bands were visualized using enhanced chemiluminescence reagents. The same membranes were re-probed with HRP conjugated GAPDH antibody (Proteintech), mouse anti-alpha tubulin antibody (Santa Cruz Biotechnology) or mouse anti-PCNA (Santa Cruz Biotechnology) to confirm equal loading of proteins for each sample.

After treatment with 5× SDS-PAGE loading buffer (Beyotime Biotechnology; Cat# P0015) at 98 °C for 10 min, samples were resolved on 8%, 10% or 12% SDS polyacrylamide gels from the SDS-PAGE preparation kit (Sangon Biotech, Shanghai, China; Cat# C631100) and electroblotted onto polyvinylidene fluoride western blot membranes (Merck Millipore KGaA, Darmstadt, Germany; Cat# 03010040001). Membranes were incubated in BeyoECL Moon detection reagent (Beyotime Biotechnology; Cat# P0018FS) and visualized using the Amersham Imager 600 (AI600 RGB; GE Healthcare, Uppsala, Sweden) after application of primary rabbit anti-Flag antibody (Sigma-Aldrich; Cat# F7425), rabbit anti-HA antibody (Cell Signaling Technology, Danvers, MA, USA; Cat# 3724; lot# 10), rabbit anti-eIF4G antibody (Cell Signaling Technology; Cat# 2498; lot# 4), rabbit anti-eIF4E antibody (Cell Signaling Technology; Cat# 2067; lot# 8), rabbit anti-GAPDH (6C5) antibody (Santa Cruz Biotechnology; Cat# sc-32233; lot# L2019) or rabbit anti-Bax antibody (Cell Signaling Technology; Cat# 14796; lot# 800) and secondary HRP-labeled goat anti-rabbit IgG (Beyotime Biotechnology; Cat# A0208; lot# 110219200406).