Fast green

Eef2 Bio-Rad Laboratories)] and subjected to PAGE and electrotransfer to polyvinylidene difluoride membrane (Thermo Fisher Scientific). To verify equal loading of protein in all lanes, the membrane was dyed with FastGreen (Bio-Rad Laboratories). The membranes were blocked for 1 hour at room temperature with Odyssey Blocking Buffer (LI-COR, Cambridge, UK) and incubated overnight at 4 C with primary antibodies diluted in Tris-buffered saline and 0.1% Tween 20 (pH 7.4). Antibodies and their dilutions are detailed in Table 2.
The membranes were washed three times for 10 minutes with Tris-buffered saline and 0.1% Tween 20 (pH 7.4) and incubated for 1 hour with horseradish peroxidaseeconjugated secondary antibodies to rabbit or mouse Igs as appropriate. Following the Tris-buffered saline and 0.1% Tween 20 (pH 7.4) wash, the ImmunStar Substrate Kit (Bio-Rad Laboratories) was used to generate chemiluminescence signal. The protein bands were quantified using C-DiGit Blot Scanner and Image Studio DiGit software version 5.2.5 (LI-COR). Western blot analyses for each analyzed protein were performed at least three times. Protein-of-interest expression was normalized to GAPDH. To quantify content of puromycinlabeled proteins, Fast Green staining of total protein was used for the intensity normalization.