Anti erk1 2 pt202 py204

Immunoblotting was performed as described previously [23 (link),37 (link),38 (link)]. Briefly, treated cells were collected and washed with PBS, and then the cells were lysed by lysis buffer for 45 min. Obtained cell lysates were quantified by Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) and separated by SDS-PAGE (25–40 μg/lane). After being transferred, PVDF membranes (PerkinElmer Life Sciences, Shelton, CT, USA) were blocked with 5% skim milk and then incubated with primary antibodies overnight at 4 °C. After washing with PBST, horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratory, West Grove, PA, USA) were incubated at room temperature. The Enhanced Chemiluminescence (PerkinElmer Life Sciences) reaction was performed, and the membranes were exposed to X-ray films (Fujifilm, Tokyo, Japan). Antibodies directed against the following proteins were used in this study: The target protein was detected by primary antibodies, including anti-Cdk5 (H-291) sc-750, anti-p35 (C-19) sc-820, anti-Ret (C-19) sc-167, anti-Egr-1(588) sc-110 from Santa Cruz Biotechnology (Dallas, TX, USA). anti-Akt (#9272), anti-phospho-Akt (Ser473) (#9271) from Cell Signaling (Danvers, MA, USA), anti-ERK1 (#610030), anti-ERK1/2 (pT202/Py204) (#612358), anti-STAT3 (#610189), phospho-STAT3 (pS727) (#612542) from BD Biosciences (San Jose, CA, USA).

All cells were rinsed with PBS (pH 7.4) and lysed in RIPA Lysis buffer (Beyotime, China) supplemented with a Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific 78440) on ice for 30 min. The tissue samples were frozen solid with liquid nitrogen, ground into a powder and lysed in RIPA Lysis buffer containing the Protease and Phosphatase Inhibitor Cocktail on ice for 30 min. When necessary, sonication was used to facilitate lysis. Cell lysates or tissue homogenates were centrifuged for 10 min (12000 g, 4°C). The supernatant was collected, and the protein concentration was calculated using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The protein levels were analyzed using western blots with corresponding antibodies. The protein levels were normalized by probing the same blots with a GAPDH antibody. The antibodies were purchased from the following sources: anti-c-Src (B-12) (sc-8056, Santa Cruz Biotechnology), anti-ERK1/2 (pT202/pY204) (BD Biosciences 612359, USA), anti-ERK1 (BD Biosciences 610031, USA), anti-PKCalpha (H-7) (sc-8393, Santa Cruz Biotechnology) and anti-GAPDH (sc-365062, Santa Cruz Biotechnology). Ras activity was detected using the Ras Activation Assay Kit from Upstate-Millipore (17–218). Protein bands were analyzed using the ImageJ software.

To measure p38, extracellular‐regulated kinase (ERK), Syk and signal transducer and activator of transcription (STAT) phosphorylation, 2 × 10⁵ NK cells in 200 μl medium were stimulated as above. Non‐activated samples were included for fold‐change calculation. Cells were incubated at 37°C and fixed with 4% paraformaldehyde at specified time‐points. Fixed cells were surface‐stained with Pacific Blue‐conjugated anti‐CD56 for 45 min, permeabilized with PermBuffer III (BD Biosciences) on ice for 30 min, then stained with Alexa647‐conjugated phospho‐specific anti‐phosphorylated STAT (pSTAT)−3 (pY705), anti‐pSTAT‐4 (pY693), anti‐pSTAT‐5 (pY694), anti‐p‐p38 (pT180/Y182) or anti‐ERK1/2 (pT202/pY204) (BD Biosciences) for 1 h. Samples were run on a BDCanto II Flow cytometer and analysed with FlowJo software version 7·6·4 (TreeStar Inc., Ashland, OR, USA).