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. 2010 Aug;21(8):1275–1280. doi: 10.1681/ASN.2009121224

Figure 4.

Figure 4.

AGS3 promotes increased epithelial cell number in a Gβγ-dependent pathway. Ba and BPK epithelial cells are transduced at a multiplicity of infection of 40 with lentiviral vectors expressing shRNA (control or specifically targeted to AGS3) or GRK2ct cDNA. (A through C) For the AGS3 knockdown experiments, the two most effective AGS3 shRNAs (see Supplemental Figure 4) are used for the Western blot (A), cell proliferation assay (B), and cAMP assay (C). (A) Western blot analysis demonstrates a specific reduction in AGS3 expression after combined AGS3 shRNA knockdown (A) compared with control shRNA (C) or vehicle-treated cells (V). No off-target knockdown of AGS5/LGN or β-actin is detected using the AGS3 shRNA. Br, brain (positive control); V, vehicle; A, AGS3 shRNA; C, control (scrambled) shRNA. (B) The genetically modified Ba and BPK cells are aliquotted into 96-well plates and examined for cell number 24 hours later by CyQuant fluorescence assay. *P < 0.001, significant difference between vehicle-treated cells. (C) cAMP levels are examined by ELISA using cell lysates obtained from the genetically modified Ba and BPK cells treated with vehicle or transduced with lentiviral vectors expressing control or specific AGS3 shRNA (n = 6 to 10 samples per group). (D) BPK epithelial cells are incubated with H-89 (10 and 50 μM) for 24 hours before determination of cell numbers by CyQuant fluorescence assay. (E) BPK epithelial cells are incubated with or without Rp-adenosine-3′,5′-monophosphorothioate (20 μM), a specific PKA inhibitor, for 5 hours. Cells are collected for protein isolation to determine the expression of AGS3 by Western blot analysis. Densitometry is performed to determine the band intensities. (F) BPK epithelial cells genetically modified to overexpress GRK2ct are aliquotted into a 96-well plate; 24 hours later, the cell proliferation is measured using the CyQuant fluorescence assay and compared with vehicle-treated BPK cells. *P < 0.001, significant difference between vehicle-treated cells. Numbers of samples in each group are shown in each graph.