Nonfat dry milk

SAN tissue from WT and AC_I^–/– mice were flash frozen in liquid nitrogen for Western blotting experiments. The same amount of total protein (5 μg) was loaded in each lane. Membranes were blocked in 3% nonfat dry milk (Bio-Rad) in TBST for 1 hour (room temperature) and then incubated with primary antibodies including anti-HCN4 (1:500 dilution, APC-052, Alomone Labs), anti–β₁-AR (1:1,000 dilution, PA1-049, Thermo Fisher Scientific), anti–β2-AR (1:1,000, PA5-27083, Thermo Fisher Scientific), anti–GRK-5 (1:1,000 dilution, PA5-23189, Thermo Fisher Scientific), anti–β-arrestin-2 (1:1,000 dilution, PA1-732, Thermo Fisher Scientific), and anti-GAPDH (1:5,000, ab8245, Abcam) antibodies, all in 3% nonfat dry milk in TBST overnight at 4°C. On the next day, the membranes were incubated with conjugated secondary antibody (Abcam) for 1 hour at room temperature, and the bands were visualized using Fujifilm LAS-3000 Imager.

The cells were washed with cold PBS, harvested with a cell lifter, centrifuged and lysed with RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na deoxycholate and 0.1% SDS) supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis IN), homogenized with a pellet pestle motor (Kimble/Kontes, Vineland, NJ) and subsequently centrifuged at 13,000 x g for 15 min at 4°C. For Western blots either aliquots of culture media containing the proteins secreted by the cultured cells or aliquots of the cell lysates containing cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Blots were blocked for 1 h in Tris-buffered saline(TBS)-Tween (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% Tween 20) containing 5% nonfat dry milk (BioRad, Hercules, CA). The membranes were then incubated overnight at 4°C with mouse monoclonal α-SMA antibody (Abcam, 1:200), and GAPDH polyclonal rabbit antibody (Abcam, 1:2000) in a 5% nonfat dry milk/TBS-Tween solution. Membranes were then washed with TBS-Tween, and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, UK) diluted 3000-fold in 5% nonfat dry milk/TBS-Tween. The signals were quantified using NIH Image J software.

Frozen placenta tissue and cell samples were homogenized and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, Beyotime). The protein concentration of the supernatant was quantified using the BCA Reagent Kit (Beyotime). Equal amounts of the protein samples (20 μg) were separated by electrophoresis on a 10 or 12% sodium dodecyl sulphate–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, CA, U.S.A.). After blocking with non-fat dry milk (Yili Milk company, Inner Mongolia, China) at room temperature for 2 h, the membranes were hybridized with anti-Pax3 antibody (Dilution 1:1000, ab180754, Abcam, Cambridge, U.K.), anti-HOXD8 antibody (Dilution 1:200, ab229321, Abcam), anti-LHX3 antibody (Dilution 1:400, ab124697, Abcam), and anti-GAPDH antibody (Dilution 1:2000, SC-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. The primary antibodies were visualized by adding secondary biotin-conjugated antibodies followed by an avidin/biotin/peroxidase complex (Vectastain ABC Elite kit; Vector Laboratories lnc, Burlingame, CA, U.S.A.) and substrate (Vector NovaRED, Vectastain). Quantification was performed by densitometric analysis using Quantity One software (Bio-Rad).