Anti cx40

The thoracic aorta samples were processed as reported [9] (link). As primary antibodies, anti-Cx37 (1∶200, Santa Cruz Biotechnology, Inc, USA), anti-Cx40 (1∶400, Santa Cruz Biotechnology, Inc, USA), anti-Cx43 (1∶1000, Abcam, Cambridge, MA, USA), anti-Cx45 (1∶200, Abcam, Cambridge, MA, USA), anti NF-κB (p65) (1∶1000, Santa Cruz Biotechnology, Inc, USA), anti IκBα (1∶1000, Cell Signaling Technology, Inc, USA), anti IκBβ (1∶2000, Cell Signaling Technology, Inc, USA), and anti Phospho IκBα(Ser32) (1∶1000, Cell Signaling Technology, Inc, USA) antibody were used.

Total RNA was prepared using TRI Reagent (Sigma-Adrich, St Louis, MO), then reverse transcribed (Invitrogen, Carlsbad, CA). Quantitative real-time RT-PCR was performed using an ABI 7500 real-time system (Applied Biosystems, Foster city, CA) and TaqMan Universal Master Mix II (Applied Biosystems, CA) with TaqMan probes GJA1 (Mm00439105_m1, Thermo Fisher Scientific Inc. USA) and GJA5 (Mm00433619_s1, Thermo Fisher Scientific Inc. USA). Protein samples were separated using a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
Blots were incubated with anti-Cx40 (1:500 dilution; Santa Cruz), anti-Cx43 (1:1000 dilution; Cell Signal), and anti-phospho-Cx43 (1:1000 dilution; Cell Signal), then with horseradish peroxidase-linked secondary antibody. The immunoblots were identified with SuperSignal West Picochemiluminescent substrate (Thermo Fisher Scientific). Band intensity was calculated using ImageJ (National Institutes of Health). Intensity data from cytoplasmic and membraneous proteins in interest were normalized to α-tubulin and pan-cadherin, respectively.

P4 heart and E15.5 embryos were cut into 15–20 μm thick sections with a cryo stat. Sections were fixed with ice-cold acetone for 5 minutes, blocked with 5% normal donkey serum diluted in PBST for 1 hour at room temperature and incubated overnight with primary antibodies at 4 °C. After rinsing 3 times for 5 minutes with PBS, proper secondary antibodies (1:500) were incubated for 1 hour at room temperature. Sections were then rinsed 3 times with PBS and then mounted with DAPI-containing mounting medium (Vector Labs). The following primary antibodies were sued: anti-phosphor-histone H3 (rabbit, 1:200, Cell Signaling Technology), anti-cleaved caspase 3 (rabbit, 1:200, Cell Signaling Technology), anti-Cx40 (goat, 1:100, Santa Cruz Biotechnology), and anti-Cx43 (rabbit, 1:100, Cell Signaling Technology).