These authors contributed equally to this paper.
Prolyl hydroxylase 2: a novel regulator of β2-adrenoceptor internalization
Version of Record online: 28 NOV 2011
© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Journal of Cellular and Molecular Medicine
Volume 15, Issue 12, pages 2712–2722, December 2011
How to Cite
Yan, B., Huo, Z., Liu, Y., Lin, X., Li, J., Peng, L., Zhao, H., Zhou, Z.-N., Liang, X., Liu, Y., Zhu, W., Liang, D., Li, L., Sun, Y., Cui, J. and Chen, Y.-H. (2011), Prolyl hydroxylase 2: a novel regulator of β2-adrenoceptor internalization. Journal of Cellular and Molecular Medicine, 15: 2712–2722. doi: 10.1111/j.1582-4934.2011.01268.x
- Issue online: 28 NOV 2011
- Version of Record online: 28 NOV 2011
- Accepted manuscript online: 21 JAN 2011 09:09AM EST
- Received: August 27, 2010; Accepted: January 19, 2011
Fig. S1 Intracellular localization of 2-AR after 40 min receptor stimulation. β2-AR-293 cells were transfected with PHD2 or PHD2 siRNA or left untreated. 48 hrs after transfection, these cells were stimulated with freshly prepared isoproterenol (ISO, 10 μM, Sigma-Aldrich) for 40 min. After washing with ice-cold PBS, the cells were incubated with anti-HA antibody (for β2-AR, Santa Cruz) for 1 hr, washed, and incubated with FITC-conjugated secondary antibody (Invitrogen) for 1 hr. Then the cells were analyzed by confocal microscopy. A representative image is shown.
Fig. S2 β2-AR expression in β2-AR-293 cells after PHD2 interference. β2-AR-293 cells were transfected with PHD2 or PHD2 siRNA or left untreated for 48 hrs. β2-AR expression was detected by western blots. GAPDH was used as the loading control. A representative image is shown.
Fig. S3 HIF-1α expression in β2-AR-293 cells after different treatment. β2-AR-293 cells were transfected with PHD2 or PHD2 siRNA for 48 hrs, then stimulated with ISO (10 μM) for the indicated time. MG132 (10 μM) was added to prevent the degradation of HIF-1α. Cells cultured in 1% O2 for 48 hrs were taken as a positive control. After treatment, the expression of HIF-1α was determined by western blot. GAPDH was used as a loading control. Scores of 0, 1 or 2 indicate PHD2 knockdown, normal PHD2 expression, or PHD2 overexpression, respectively. A representative image is shown.
Fig. S4 Interaction of PHD2 with GRK protein. β2-AR-293 cells were co-transfected with His-tagged PHD2 and FLAG-tagged GRK 2, 5 or 6 plasmids for 48 hrs. The whole cell lysates were immunoprecipitated with the His antibody (for PHD2). The presence of GRKs in the immunoprecipitates was detected by western blots. A: Cells without ISO stimulation; B: Cells stimulated with ISO (10 μM) for 10 min.
Fig. S5 Estimation the expression of β2-AR protein after PHD3 interference. β2-AR-293 cells were transfected with PHD3 siRNA, scramble siRNA (Scr) or left untreated (Ctrl) for 48 hrs, and then these cells were lysed. Western blots were conducted to determine the level of β2-AR expression. GAPDH expression was used as the loading control. β2-AR expression was determined as the ratio of densitometric value compared to GAPDH expression. Representative immunoblots are shown along with quantitative data showing the mean ± S.E.M. from four separate blots.
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