Overexpression of GPR34 drives ERK1/2 activation and ERK1/2-mediated gene transcription in HeLa and lymphoma B cells. (A) HeLa cells expressing vector control, WT or DRY were analyzed for GPR34 expression by flow cytometry. (B) HeLa cells were transiently transfected with a YFP vector control or a YFP-tagged GPR34 and were visualized by confocal microscopy. (C) HeLa vector control, WT, or DRY cells were analyzed for total and phosphorylated forms of ERK1/2 with and without PMA activation by Western blot. (D) Average fold increase in ERK1/2 phosphorylation. Data are normalized to total ERK1/2. *Compared with GPR34-DRY, GPR34 WT cells had a significant increase in ERK1/2 phosphorylation (P = .05, n = 5). (E) HeLa vector control or WT cells were transiently transfected with reporter gene plasmids that have the firefly luciferase gene under the control of a specific cis-acting; pNFAT-luc, pISRE-luc, pSRE-luc, pE2F-luc, pNFKB-luc, pCRE-luc, or pAP1-luc. Data represent an average fold increase in luciferase activity over vector control (n = 3). (F) Activation of the pAP1 and pCRE reporters in vector control, WT, and DRY cells in the absence or presence of increasing doses (25-100μM) of the MEK inhibitor PD98059 (n = 2). *Compared with the nil control, WT cells have a significant increase in pAP1 (P = .04) and pCRE (P = .002) activity. (G) OCI-Ly19 vector control or WT cells were analyzed for total and phosphorylated forms of ERK1/2, PKC, and CREB Western blot.