Phospho stat5 tyr694 d47e7

Hela cells were lysated after 48 h transfection with pRRL-PGK-T2AGFP–STAT5B WT and mutant constructs. Non-transfected cell line and cell transfected with an empty vector were used as internal control for the reaction. Standard western blot analysis was carried out. RIPA buffer supplemented with protease inhibitors cocktail (Sigma Aldrich) and phosphates inhibitor cocktail (Sigma Aldrich) was used for protein extraction. Proteins were transferred to a polyvinylidene difluoride membrane, after which the membrane was blocked with Super Block (Thermo Fisher) blocking buffer for 1 h. Primary antibodies for STAT5B were obtained from Cell Signal Technology: STAT5B Rabbit mAb#9363 dilution 1:1,000 in 5% milk and Phospho-Stat5 (Tyr694) (D47E7) Rabbit mAb #4322 dilution 1:500 in Super Block. Monoclonal Anti-Actin (Clone AC-40) and anti-GFP (clone GFP-20) antibodies were purchased from Sigma Aldrich and used to a working dilution of 1:1,000. Membranes were incubated overnight at +4 °C. Uncropped representative WB images are shown in Supplementary Fig. 11.

Sample preparation of whole cell lysates, SDS-PAGE, membrane transfer and blotting were performed according to standard protocols. Antibodies to p21 (#2947), PTPN2 (TCPTP) (#58935), Jak2 (D2E12) (#3230), Phospho-Jak2 (Tyr1007/1008) (#3771), Stat3 (D1B2J) (#30835), phospho-Stat3 (Tyr705) (D3A7) (#9145), Stat5 (D3N2B) (#25656), phospho-Stat5 (Tyr694) (D47E7) (#4322), MDM2 (D1V2Z) (#86934), α-Tubulin (#2144) and GAPDH (#5174) were purchased from Cell Signaling Technologies. Antibody to TP53 (sc-47698) and MDMX (G-10) (sc-74467) were purchased from Santa Cruz Biotechnology. Antibodies were used at 1:1000 dilution (Cell Signaling Technologies) or at 1:500 dilution (Santa Cruz Biotechnology).
Uncropped western blots from the main figures are provided in Supplementary Figure 8.