SMAD1 Polyclonal Antibody

Protocol tips

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	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

rChrdl1 was titrated to identify the concentration that maximally impaired BMP4 and BMP7 (50 ng/ml) (catalog number 120-03; Peprotech). Total protein (30 to 40 μg) was separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and resolved proteins transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h with 5% milk solution, except for membranes used to detect pSmad1/5/8, total Smad2/3, and ShcA, for which 5% bovine serum albumin (BSA) solution was used as the blocking agent. Membranes were washed with 1× Tris-buffered saline (TBS)–1% Tween 20 and incubated overnight at 4°C using the following antibodies, as indicated: ShcA (1:1,000 dilution) (catalog number 610081; BD Biosciences), FLAG (M2) (1:1,000 dilution) (catalog number F1804; Sigma), ErbB2/Neu (C-18) (1:1,000 dilution) (catalog number sc-284-G; Santa Cruz), phospho-Smad 1/5/8 (1:1,000 dilution) (catalog number 9511; Cell Signaling), phospho-Smad1/8 (1:1,000 dilution) (catalog number 562508; BD Biosciences), Smad1 (1:1,000 dilution) (catalog number 38-5400; Invitrogen), Smad2/3 (1:1,000 dilution) (catalog number 610842; BD Biosciences), phospho-Smad2 (Ser465/467) (1:1,000 dilution) (catalog number 3101; Cell Signaling), and α-tubulin (1:5,000 dilution) (catalog number T9026; Sigma). Membranes were washed 3 times with 1× TBS–1% Tween 20 and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution) for 1 h. The membranes were washed 3 times with 1× TBS–1% Tween 20 and visualized with an enhanced chemiluminescence system.

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