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. Author manuscript; available in PMC: 2019 Jul 23.
Published in final edited form as: AAPS J. 2019 Feb 13;21(2):30. doi: 10.1208/s12248-019-0303-4

Fig. 7.

Fig. 7.

(a) Effects of SUMO1, SUMO2, SUMO3 and Ubc9 on hOAT3 SUMOylation. Top panel: cDNAs for HA-tagged SUMO1, SUMO2, or SUMO3 were transfected into COS-7 cells separately with 2.4μg of Ubc9, a SUMO-conjugating enzyme. 48 hrs after transfection, hOAT3 was pulled down by anti-Myc antibody (hOAT3 was tagged with the Myc epitope), with subsequent immunoblotting (IB) using anti-HA antibody. Bottom panel: The same blot from the top panel was re-probed with anti-Myc antibody to detect the amount of hOAT3 pulled down. (b) Effects of Ubc9 on hOAT3 SUMOylation. Top panel: cDNAs for HA-tagged SUMO2 was transfected into COS-7 cells with different amount of Ubc9 for 48 hrs. After transfection, hOAT3 was pulled down by anti-Myc antibody (hOAT3 was tagged with the Myc epitope), with subsequent immunoblotting (IB) using anti-HA antibody. Bottom panel: The same blot from the top panel was re-probed with anti-Myc antibody to detect the amount of hOAT3 pulled down. (c) PKA Specificity on hOAT3 SUMOylation. Top panel: hOAT3-expressing cells were transfected with HA-SUMO2 and 2.4μg of Ubc9 for 48h, then pretreated with or without H-89 (4μM, 10min). After that, cells were treated with the Bt2-cAMP (10μM, 30min) in the presence and absence of PKA inhibitor H-89 (4μM, 30min). hOAT3 was pulled down by anti-Myc antibody, with subsequent immunoblotting (IB) using anti-HA antibody. Bottom panel: The identical blot from the top panel was re-probed with anti-Myc antibody. (d) Densitometry analyses of results from Fig. 7c. Values are mean ± S.D. (n = 3). *P<0.05.