FIGURE 3.

Knockdown of Sh3rf2 promotes JNK and c-Jun activation in a POSH-dependent fashion. A, HEK293 cells were transfected with a siRNA targeting human Sh3rf2 (sihuSh3rf2) or scrambled siRNA. Lysates were collected 20 h later and subjected to immunoblotting using an antibody specific for phospho-JNK. Membranes were then reprobed for total JNK. B, HEK293 cells were treated as above and lysates were subjected to immunoblot using an antibody specific for phospho-c-Jun. Membranes were reprobed with an antibody detecting total c-Jun. C and D, HEK293 cells constitutively expressing siRNA targeting POSH (siPOSH cells) were treated as in A and B, respectively. Note the lack of JNK and c-Jun activation in these cells. E, primed PC12 cells were co-transfected with pCMS-EGFP and the indicated constructs in pCMV. Cells were transfected with the indicated synthetic siRNAs and transfected (EGFP+) cells were counted beginning 24 h later (Day 1). Shown is one experiment performed in triplicate. The experiment was performed three times with similar results (*, p < 0.01 relative to same time point for siSh3rf2 with dominant-negative c-Jun and siSCRAM with empty vector).