Anti p smad2 s465 467

Proteins were seperated on a 7.5% Bisacrylamide gel, and transferred to a nitrocellulose membrane using wet transfer (Towbin buffer, 2.5 h 275 mA at 4 °C). After overnight incubation at 4 °C with 1:1000 polyclonal Rabbit anti P-Smad1/5 (S463/465)/Smad8 (S426/428) (Cell signaling, USA) or anti P-Smad2 (S465/467) (Cell signaling, USA), membranes were incubated with 1:1500 polyclonal Goat anti Rabbit labeled with horseradish peroxidase (HRP) (DAKO, Belgium) for 1 h. Hereafter, enhanced chemiluminescence using ECL plus (GE Healthcare, UK) was used to visualize the proteins. To visualize overexpression of constitutively active ALKs, a rabbit polyclonal antibody directed against their internal HA tag was used: HA-probe Antibody (Y-11) (Santa Cruz, USA) (1:1000). As loading control either Gapdh was stained with an anti-Gapdh mouse mAb (1G5) (Sigma Aldrich, Germany) (1:10 000) in combination with IRDye 680RD Donkey anti mouse (1:10 000) (Licor, USA) using the Odyssey detection system (Licor, USA) or Vinculin with a rabbit pAb (H300) (Santa Cruz, USA) (1:1000) in combination with HRP-labeled Goat-anti Rabbit (1:2000) (DAKO, Denmark) using ECL. Finally, blots were quantified using ImageJ.

Specific antibodies were purchased from the following commercial sources: Anti-AFP, anti-ALB, anti-CD44, anti-Evi1, anti-flag (mouse), anti-HA, anti-HNF4α, anti-H3, anti-H3K36me3, anti-Myc, anti-OCT4A, anti-P300, anti-PPM1A (rabbit), anti-pSmad2 (S465/467), anti-pSmad2 (S245/250/255), anti-pSmad3 (s423/425), anti-Smad2, anti-Smad3 (rabbit), anti-SnoN, anti-Sox2, anti-TAT, and normal rabbit IgG from Cell Signaling Technology (Danvers, MA); anti-PPM1A (mouse), anti-SC35, and anti-SETD2 were from Abcam (Cambridge, MA); Anti-Smad4 and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); Anti-β-actin, and anti-flag (rabbit) from Sigma-Aldrich (St. Louis, MO); Alexa594 goat anti-mouse IgG from Life Technology (Carlsbad, CA); Dylight488 goat anti-rabbit IgG from Vector Labs (Burlingame, CA).

Equal amounts of the total protein isolated from murine lungs, primary cultured fibroblasts, or HEK293 cells were prepared in radioimmunoprecipitation assay buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% Nonidet-P40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), and supplemented with 1 mM dithiothreitol (DTT), 100 nM MG132, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Nacalai Tesque). For western blot analysis, equal amounts of total protein (10–15 μg) from the lysate were subjected to SDS-polyacrylamide gel electrophoresis. The primary antibodies used in the present study were anti-PAF-AH2 (TI10; 1:1000), anti-β-Actin (sc-47778; Santa Cruz Biotechnology; 1:1000), anti-p-Smad 2 S465/467 (#3108), and anti-Smad 2 (#5339) (Cell Signaling Technology; 1:1000). Protein bands were visualized using horseradish peroxidase-conjugated secondary antibodies (1:2000) and enhanced chemiluminescence (Chemi-Lumi One Super; Nacalai Tesque) on a LAS-4000 mini (GE healthcare). Protein bands were quantified using ImageJ ver1.52.