Phospho-SMAD2 (Ser465, Ser467) Monoclonal Antibody (H.205.4)

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	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Cells were lysed in RIPA buffer (Pierce, Rockford, IL) on ice. The samples were denatured at 95 °C for 5 min in sample buffer containing 2% SDS and 1% 2-mercaptoethanol, separated on 10 or 15% SDS–polyacrylamide gels, and transferred to polyvinylidene difluoride membranes by a semi-dry transfer apparatus (Bio-Rad, Hercules, CA). For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C. The proteins were then transferred to polyvinylidene difluoride membranes at 4 °C by a wet transfer apparatus (Bio-Rad, Hercules, CA). The membranes were blotted with 5% milk for 1 h and then incubated with primary antibodies overnight. The following antibodies were used: mouse anti-GDF11 (0.5 μg ml−1, MAB19581, clone #743833, R&D), rabbit anti-phospho-Smad2 (1:1,000, MA5-15122, l, ThermoFisher), rabbit anti-phospho-Smad3 (1:1,000, #9520, Cell Signaling), rabbit anti-phospho-Smad2/3 (1:1,000, #8828, Cell Signaling), rabbit anti-Smad2/3 (1:1,000, #8685, Cell Signaling), rabbit anti-phospho-c-Fos (1:1,000, #5348, Cell Signaling), rabbit anti-c-Fos (1:1,000, #2250, Cell Signaling), mouse anti-NFATc1 (1:2,000, MA3-024, ThermoFisher), rabbit anti-phospho-Smad1/5 (1:1,000, #9516, Cell Signaling), and rabbit anti-Smad1/5/8 (1:1,000, SC-6031-R, Santa Cruz). The immunocomplexes were incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and visualized with SuperSignal reagents (Pierce, Rockford, IL). The ImageJ software was used for densitometric analyses of the blots, and the quantification results were normalized to the loading control.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Phospho-SMAD2 (Ser465, Ser467) Monoclonal Antibody (H.205.4) below.

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