Supplementary Information
Protein kinase A and p38-MAP kinase crosstalk contributes to regulating
aquaporin-2 abundance
Pavel I. Nedvetsky1*, Vedrana Tabor1*, Grazia Tamma2, Sven Beulshausen1,Philipp Skroblin1, Aline Kirschner1, Kerim Mutig3,Mareike Boltzen1, Oscar Petrucci1, Anna Vossenkämper1, Burkhard Wiesner1, Sebastian Bachmann3, Walter Rosenthal4,5, Enno Klussmann1
1Leibniz-Institute for Molecular Pharmacology, Berlin, Germany; 2University of Bari, Department of General and Environmental Physiology, Bari, Italy; 3Center for Anatomy, Charité-Humboldt University, Berlin, Germany; 4Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; 5 Department of Molecular Pharmacology and Cell Biology, Charité–University Medicine Berlin, Berlin, Germany
*These authors contributed equally to this paper
Running head: PKA and p38MAPK control AQP2 protein levels
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The forskolin-induced increase in the level of AQP2 is detectable with different anti-AQP2 antibodies.
To rule out that detection of FSK-induced increases in AQP2 signals are due to altered recognition of the antigen by the antibody used, we compared following antibodies: H27, a rabbit antiserum directed against the C-terminus of AQP2 generated in our laboratory;1 C-17, a goat antibody available from Santa Cruz Biotechnology (Cat. #: sc-9882) directed against the C-terminus of AQP2, and N-20, a goat antibody from Santa Cruz Biotechnology (Cat. #: sc-9880) directed against the N-terminus of AQP2.
HEK293 cells were transfected with plasmids encoding wild-type AQP2 and were left untreated or after 24 hours were treated with FSK (10 μM, 60 min). AQP2 was detected by Western blotting with either C-17 (Suppl. Fig. 1A) or N-20 (Suppl. Fig. 1B) antibody. In both cases an increase in AQP2 signals was observed after treatment with FSK. Based on the observation that two antibodies directed against different epitopes located in distinct regions of the protein yielded similar results, we conclude that the increases in AQP2 signals observed are unlikely to be due to changed immunogenicity after treatment with forskolin but reflect increases in AQP2 protein levels.
Legend to suppl. Figures
Suppl. Figure 1. The forskolin-induced increase in the level of AQP2 is detectable with different anti-AQP2 antibodies. HEK293 cells were transiently transfected with plasmids encoding wild-type AQP2. The cells were lysedand AQP2 was detected with the indicated anti-AQP2 antibodies by Western blotting. A. antibody C-17, directed against the C terminus of AQP2 and B. antibody N-20, directed against the N terminus of AQP2; AQP2-compl, complex glycosylated AQP2; AQP2-hm; high mannose glycosylated form of AQP2; AQP2-ng, non-glycosylated AQP2; FSK, forskolin; WB, Western blot. For details see suppl. Information.
Suppl. Figure 2. dbcAMP rapidly increases the AQP2 protein level in IMCD cells. A. IMCD cells were left untreated or treated with dbcAMP (500 µM, 60 min), lysed, and AQP2 was immunoprecipitated and detected by Western blotting. Shown is a representative blot of three independent experiments. B. Semiquantitative analysis of the effect of dbcAMP on the AQP2 protein level. Western blots obtained as indicated in A were densitometrically analyzed. Shown are means ± SEM of three independent experiments each performed in duplicate. Statistically significant differences vs. untreated cells are indicated: *, p < 0.05.
Suppl. Figure 3. Inhibition of p38 MAPK with SB202190, and forskolin decrease cyclin D3 phosphorylation (p-cyclinD3). IMCD cells remained untreated or were treated with forskolin (FSK; 10µM, 30 min) or SB202190 (5µM, 120 min). AQP2 was detected by Western blotting. Shown is a representative blot of two independent experiments.
Reference
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