P stat5 y694

Total proteins in spleens or aortas were extracted via High Efficiency RIPA Lysis Buffer (KeyGEN BioTECH), quantified by BCA protein concentration assay kit (Beyotime Biotechnology), and denatured in boiling water with SDS-PAGE protein loading buffer (Beyotime Biotechnology). 30 μg of proteins per sample was loaded in 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as blocking agent before antibody incubation. Polyvinylidene fluoride (PVDF) membranes were incubated with rabbit antibodies against p-STAT3-Y705 (Abcam), p-STAT5-Y694 (Cell Signaling Technology), SOCS3 (Abcam), FOXP3 (Abcam), β-ACTIN (Abways), and Goat anti-rabbit IgG with HRP (Biosharp), respectively. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Blot data were analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas are measured, and the relative expression amount of the protein samples is calculated by the method of the target protein gray value/internal reference β-ACTIN gray value.

Cells were lysed with radioimmunoprecipitation buffer (50 mM Tris-HCl pH-7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitor mixture, 2 mM Na₃VO₄, 10 mM NaF and 1 mM PMSF on ice. Cell lysates were cleared by centrifugation and quantified using the bicinchoninic acid method. Proteins (10-40 μg) were separated by SDS–PAGE under reducing conditions and blotted onto a PVDF membrane (Millipore). Membranes were probed with specific antibodies. Blots were washed and probed with respective secondary peroxidase-conjugated antibodies, and the bands visualized by chemiluminescence. The following antibodies were used: mouse monoclonal antibodies for FLAG-Tag (1:3000, Sigma), β-Actin (1:5000, Sigma), phospho-Tyrosine (1:1000, Cell Signaling), t-FAK (1:200, Santacruz Biotech), rabbit monoclonal antibodies for HA-Tag (1:2000, Cell Signaling), JAK2 (1:1000, Cell Signaling), pJAK2-Y1007/1008 (1:1000, Cell Signaling), pSTAT3-Y705 (1:1000, Cell Signaling), pSTAT5-Y694 (1:1000, Cell Signaling), GAPDH (1:1000, Cell Signaling), SP1 (1:1000, Cell Signaling), pFAK-Y925 (1:1000, Cell Signaling), rabbit polyclonal antibodies for Histone H3 (1:1000, Abcam).

To evaluate protein expression, cells were lysed in buffer (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 5 mM EDTA; 1% (v/v) NP-40, 1 mM Na₃VO₄; 10 mM NaF; 10 mM NaPyrophosphate; supplemented with protease inhibitor cocktail Complete Mini (Roche)), supplemented with 1 mM of AEBSF (Bio-Rad). Total protein was quantified using the Bradford assay (Bio-Rad). Before resolving the protein extracts, samples were resuspended in Laemmli sample buffer (Bio-Rad 1610737) and denatured for 5 min at 95 °C. Equal amounts of protein extracts were resolved by 12% SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with the following primary antibodies: p-STAT5 (Y694) (#9359), p-Akt (S473) (#9271), p-Erk (T202/Y204) (#9101), p-S6 (S235/236) (#2211), STAT5 (#94205), Akt (#9272), Erk (#4695) and S6 (#2217) (all from Cell Signaling Technology) and β-actin (sc-47778 from Santa Cruz Biotechnology). Immunodetection was performed by incubation with horseradish peroxidase-conjugated secondary antibodies (Promega Corporation) and developed by chemiluminescence detection using the Pierce^TM ECL Western Blotting Substrate (ThermoFisher). Films exposed to the membranes were developed in a Curix60 (AGFA HealthCare). Amersham^TM ECL^TM Rainbow^TM Marker – Full range (GE Healthcare) was used as molecular weight reference.

Fresh purified NK cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo). Then, samples were run on precast 4–12% Bis–Tris protein gels (Genscript). Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% w/v skimmed milk at room temperature for 1 h. Then, PVDF membranes were incubated with primary antibodies in 5% w/v skimmed milk in Tris-buffered saline containing 0.1% Tween-20 at 4 °C overnight, then incubated with Anti-rabbit IgG, HRP-linked Ab (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. To detect several proteins on the same PVDF membrane, membranes were processed with western stripping buffer (Beyotime) and reincubated with primary Abs. Chemiluminescence autoradiography was used to detect protein bands. The primary Abs for METTL3, WTAP, YTHDC2, YTHDC1, YTHDF1, ALKBH5, FTO, SHP2, AKT, p-AKT S473, STAT5, p-STAT5 Y694, Lamin B1, mTOR, p-mTOR Ser2448, p38 MAPK, p-p38 MAPK Thr180/Tyr182, and β-actin were purchased from Cell Signaling Technology; primary Ab SOCS3 was purchased from Abcam. The Anti-rabbit IgG, HRP-linker antibody was purchased from Cell Signaling Technology. The dilution of all antibodies used for immunoblotting was 1:1000.

Cell lysates were resolved by 10% SDS-PAGE (Invitrogen), protein was transferred onto nitrocellulose membranes and immunoblotted with antibodies against p-STAT5 (Y694) (1:1000), STAT5 (1:1000) (Cell Signaling Technology), BCL-2 (1:5000) (Santa Cruz), or β-actin (1:20000) (Sigma). Immunodetection was performed by incubation with SuperSignal West Dura Substrate (ThermoFisher).