Eef1b2

For western blotting, 20 μg protein sample was loaded into each lane of a 4 to 12% Bis-Tris polyacrylamide gel followed by a transfer to a polyvinylidene difluoride membrane. Total protein on the membrane was measured a No-Stain Protein Labeling Reagent (Invitrogen) and quantified using ImageJ. Membranes then were blocked using 5% milk in tris-buffered saline (TBS) for 1 h and followed with incubation overnight with following primary antibodies in 5% bovine serum albumin [Puromycin 1:2,000 (Millipore), eEF1A 1:1,000 (clone CBP-KK1 Millipore) GAPDH 1:1,000 (Cell Signaling Technologies), eEF1A2 1:1,000 (Abcam) Pan-Actin 1:1,000 (Sigma), GAPDH 1:1,000 (Cell Signaling Technologies), eEF1B2 1:1,000 (Proteintech), eEF1D 1:1,000 (Abnova), and eEF1G 1:1,000, (Santa Cruz Biotechnology)]. Membranes then were washed three times for 5 min each with TBS 0.2% Tween and followed by incubation with horseradish peroxidase-conjugated secondary antibodies in 5% dry milk for 1 h. Immunoreactive bands were detected using chemiluminescence (Cytiva) using the ProteinSimple imaging system (Bio-Techne). Band intensities were quantified using ImageJ.