Inactivation of FAM20B inhibited the activation of TGFβ signaling pathway in AF. (A, B, C, D) IHC Detection of P-Smad 2 and P-Smad 3 in IVDs of 3-week-old control and cKO mice (representative images of at least 3 mice for each group). A1, B1, C1, D1 were the higher magnification views of the black boxes in A, B, C, D, respectively. A2, B2, C2, D2 were the higher magnification views of the black boxes in A1, B1, C1, D1, respectively. The numbers of P-Smad 2 or P-Smad 3 positive cells were reduced in the AF of cKO mice (Bars in the A, B, C, D = 200 μm, Bars in the A1, B1, C1, D1 = 50 μm, Bars in the A2, B2, C2, D2 = 10 μm). (E) Protein levels of P-Smad 2 and P-Smad 3 in AF as revealed by Western immunoblotting. (F) Relative levels of P-Smad 2 and P-Smad 3 to total P-Smad 2/3 determined by Western immunoblotting (n = 3; * = p < 0.05). The protein levels of both P-Smad 2 and P-Smad 3 were significantly lower in the AF of cKO mice. For Western immunoblotting analyses, the total proteins were extracted from AF of 3-week-old control and cKO mice.