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. 2017 Apr 13;292(23):9523–9539. doi: 10.1074/jbc.M116.771394

Figure 2.

Figure 2.

PKD1 phosphorylation at Ser203 can be dissociated from PKD or PKC family catalytic activity. A and B, confluent cultures of IEC-18 cells were incubated in the absence (0) or in the presence of increasing concentrations of CRT0066101 (A) or kb NB 142-70 (B) for 1 h and then stimulated without (−) or with 10 nm ANG II for 10 min. Then, the cultures were lysed with 2×SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect PKD1 phosphorylated at either Ser203 (pSer203) or Ser916 (pSer916). Lysates were also analyzed for total PKD1 (PKD). C, quantification of three independent experiments similar to those shown in A and B are included: open circles represent Ser(P)203, whereas closed circles signify Ser(P)916. D and E, confluent cultures of Swiss 3T3.PKD1 cells were incubated in the absence (0) or in the presence of increasing concentrations of CRT0066101 (D) or kb NB 142-70 (E) for 1 h and then stimulated without (−) or with 10 nm bombesin for 10 min. Then the cultures were lysed with 2×SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect PKD1 phosphorylated at either Ser203 (pSer203) or Ser916 (pSer916). Lysates were also analyzed for total PKD1 (PKD). F, quantification of three independent experiments similar to those shown in D and E are included: open circles represent Ser(P)203, whereas closed circles signify Ser(P)916. G, Swiss 3T3.PKD1618N cells were stimulated with bombesin (Bom) for the indicated times. Then the cultures were lysed with 2×SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect PKD1 phosphorylated at Ser203 (pSer203) and GAPDH. H and J, confluent cultures of IEC-18 cells (H) or Swiss 3T3 cells (J) were incubated in the absence (0) or in the presence of increasing concentrations of Go6983 or 3.5 μm GFI (GF) for 1 h as indicated and then stimulated without (−) or with 10 nm ANG II (H) or 10 nm bombesin (J) for 10 min. Then the cultures were lysed with 2×SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect PKD1 phosphorylated at either Ser203 (pSer203) or Ser744 (pSer744) as a marker of PKC activity. Lysates were also analyzed for total PKD1 (PKD).