Figure 3Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in HeLa cells transfected with Nedd4l siRNA (siNedd4l) or control RNA (siNC) (A) , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in Nedd4l+/+ and Nedd4l-/- MEFs , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.Immunoblot analysis of phosphorylated p38 (P-P38), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, NEDD4L, and actin in Nedd4l+/+ and Nedd4l-/- primary lung epithelial cells (C), or Nedd4l+/+ and Nedd4l+/- astrocytes (D) , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.(E) Immunoblot analysis of phosphorylated p65 subunit of NF-κB (P-P65), total P65, phosphorylated p38 (P-P38), total P38, MEKK2, NEDD4L, and actin in HeLa cells transfected with Myc-tagged NEDD4L，Myc- tagged NEDD4L-CA(C942A), or Myc- tagged Null plasmids, treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.(F)Immunoblot analysis of MEKK2, TRAF2, TRAF6, NEDD4L, and actin in Nedd4l+/+, Nedd4l+/-, and Nedd4l-/- MEFs. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.(G)Immunoblot analysis of MEKK2 in lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with CHX (50 μg/ ml) for indicated time. (H)The intensity values were measured by Image J software to calculate half-life of MEKK protein ( n = 3 biological replicates). Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown. Significant differences were tested using a two-tailed Student's t-test. *P<0.05, **P<0.01. NS, no significance.