Figure 3. Effect of PRR siRNA transfection and bafilomycin A1 treatment on intracellular GH accumulation and GH release in GH3 cells.

a GH3 cells were transfected with either scramble control siRNA (control) or PRR si-RNA (si-PRR) for 48 h, and cell lysates were subjected to immunoblotting using anti-GH, anti-PRR, anti-PLZF, and anti-actin antibodies. b Conditioned medium from cultured GH3 cells transfected with either scramble control siRNA (control) or PRR si-RNA (si-PRR) for 48 h was extracted and immunoblotted with an anti-GH antibody. A representative immunoblot (lower panel) and quantification of GH protein levels normalized to WST-1 absorbance (n = 3, upper panel) are shown. **P < 0.01. c GH3 cells were treated without (control) or with bafilomycin A1 (BafA1, 100 nM) for 24 h, and cell lysates were subjected to immunoblotting using anti-GH, anti-PRR, anti-PLZF, and anti-actin antibodies. d Conditioned medium from cultured GH3 cells treated without (control) or with bafilomycin A1 (BafA1, 100 nM) for 24 h, and cell lysates were subjected to immunoblotting using an anti-GH antibody. A representative immunoblot (lower panel) and quantification of GH protein levels normalized to WST-1 absorbance (n = 3, upper panel) are shown. *P < 0.05. e GH3 cells were transfected with either scramble control siRNA (control) or PRR si-RNA (si-PRR) for 48 h, then treated without or with bafilomycin A1 (BafA1, 100 nM) for 9 h, and cell lysates were subjected to immunoblotting using anti-GH and anti-actin antibodies. f GH3 cells were treated without (control) or with recombinant human prorenin (5 nM) for 24 h, and cell lysates were subjected to immunoblotting using anti-GH and anti-actin antibodies. g Subconfluent GH3 cells were replaced with serum-free medium without (control) or with prorenin (5 nM), incubated for the indicated times and GH levels in each conditioned media were measured by GH ELISA (n = 4). Each column and bar represents mean ± SEM. Full-length blots were shown in supplementary figures 1 and 2.