sentence: The membranes were probed overnight at 4 ° C with the following antibodies : ALK5 ( TGF β R ‐ I , 1:1000 ; Cell Signaling Technology , Danvers , MA , USA ) , Phospho ‐ Smad2 ( Ser465 / 467 ) / Smad3 ( Ser423 / 425 ) ; 1:1000 ; Cell Signaling Technology ) , Phospho ‐ SMAD1 / 5 ( Ser ‐ 463/465 ; 1:1000 ; Cell Signaling Technology ) , PAI ‐ 1 ( 1:1000 ; Santa Cruz Biotechnology , Dallas , TX , USA ) or β ‐ actin ( 1:1000 , Santa Cruz Biotechnology ) .