Anti rabbit igg from donkey

Protein samples were resolved by SDS–polyacrylamide gel electrophoresis on NuPAGE Novex 4–12% Bis-Tris gels (NP0321 and WG1402A, ThermoFisher Scientific) with NuPAGE MOPS SDS buffer (NP0001, ThermoFisher Scientific), or NuPAGE Novex 3–8% Tris-Acetate gels (EA0375BOX and WG1602BOX, ThermoFisher Scientific) with NuPAGE Tris-Acetate SDS buffer (LA0041, ThermoFisher Scientific). Resolved proteins were either stained with colloidal Coomassie blue dye (‘Instant Blue’, Expedion), or were transferred onto a nitrocellulose iBlot membrane (Invitrogen) with the iBlot Dry Transfer System (Invitrogen).
Antibodies used for protein detection in this study are described in Appendix 1-key resources table. Conjugates to horseradish peroxidase of anti-sheep IgG from donkey (Sigma, A3415), anti-rabbit IgG from donkey (GE Healthcare, NA934), or anti-goat IgG from rabbit (Sigma, A5420) were used as secondary antibodies before the detection of chemoluminescent signals on Hyperfilm ECL (Amersham, GE Healthcare) using ECL Western Blotting Detection Reagent (GE Healthcare).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed using NuPAGE Novex 4–12% Bis-Tris precast gels (Invitrogen). Western blotting was performed according to standard protocol using a nitrocellulose (Whatman, Protran) or PVDF (Millipore, Immobilon-P) membrane. The following antibodies were used: phospho-RPS6 (Ser235/236), phospho-4EBP1 (Thr37/46) (Life Technologies, Grand Island, NY, USA), p53, p27 (AnaSpec, Inc.), S6 (Santa Cruz Biotechnology, USA) and α-tubulin (Sigma, MO, USA). Secondary antibodies were HRP linked, anti-rabbit IgG (from donkey) and anti-mouse IgG (from sheep) (GE Healthcare, NA934V, NA931V and NA935V, respectively).