Journal: Cellular signalling

Article Title: Distinct bone morphogenetic proteins activate indistinguishable transcriptional responses in nephron epithelia including Notch target genes.

doi: 10.1016/j.cellsig.2011.09.008

Figure Lengend Snippet: Fig. 4. BMP signaling directly activates a set of typical Notch response genes. A: QPCR confirming that HES1, HEY1, SNAI1 and SNAI2 are strongly up-regulated in RPTECs after treat- ment for 2 or 6 h with 50 ng/ml of BMP2 (left panel) and BMP7 (right panel). The less durable response of BMP7 at 6 h may result from increased amounts of secreted CHRDL1 accumulating in the medium and resulting in greater antagonism of BMP7, but not BMP2. B: RPTECs were incubated for 30 min with cycloheximide (5 μM) or vehicle prior to the addition of BMP7 (50 ng/ml) for 2 h and analyzed by RT-qPCR. Cycloheximide did not reduce target gene activation showing that protein synthesis is not required for BMP7 induction of expression. C: Cells were incubated with vehicle or 5 μM DAPT for 30 min prior to adding BMP7 at 50 ng/ml. QPCR shows that the inhibition of Notch signaling did not significantly reduce gene induction at 2 h. D: RPTECs were serum starved for 2 h, then treated for the time specified with BMP2 at 50 and 100 ng/ml or with BMP7 at 100 ng/ml. Immunoblots were probed for SNAI1 (Snail) and SNAI2 (Slug). Blots for Beta tubulin below each protein of interest show relative loading of sample. E: QPCR showing BMP2 expression in RPTECs transduced for 32 h with adenoviral vectors expressing GFP (control), HEY1 or SNAI1. F: QPCR showing reduction in BMP2 expression in RPTECs incu- bated with 50 ng/ml of BMP2 for 2, 6, and 24 h. Asterisk indicates p valueb0.05.

Article Snippet: Analysis was performed as previously described [23], using rabbit antibodies against PhosphoSmads 1, 5, and 8 (Cell Signaling Technology), SNAI2 (ProSci), Beta Tubulin (Santa Cruz Biotechnology) and mouse antibodies against BMP2 (Abcam) and SNAI1 (Origene Technologies) overnight at 4°.

Techniques: Incubation, Quantitative RT-PCR, Activation Assay, Expressing, Inhibition, Western Blot, Control