Figure 3. Fam20A enhances Fam20C-dependent phosphorylation.

(A and B) Effect of Fam20A KO on the phosphorylation of AMBN (A) or OPN (B). C-terminal V5-tagged AMBN or OPN (AMBN-V5 or OPN-V5) was expressed in WT or Fam20A-KO ALCs and analyzed as in Figure 1C. Flag-tagged Fam20A (20A-Flag) was co-expressed in Fam20A-KO ALCs as indicated. (C) Phosphorylation of OPN-V5 in WT, Fam20A-KO, Fam20C-KO, or Fam20A and Fam20C double-KO (A&C-DKO) ALCs. The experiment was performed as in (B). Two different exposures of autoradiography are shown. (D–F) Effect of Fam20A overexpression on the phosphorylation of OPN (D), ENAM (173–277) (E) or AMBN (F) in WT or Fam20C-KO U2OS cells. C-terminal V5-tagged OPN, ENAM (173–277) or AMBN (OPN-V5, ENAM-V5 or AMBN-V5) were expressed in WT or Fam20C-KO U2OS cells and analyzed as in Figure 1C. Fam20A-Flag was stably expressed as indicated. Fam20A-Flag in the cell lysates and endogenous Fam20C in the conditioned medium are also shown. Fam20C-KO U2OS cells were generated by means of transcription activator-like effector nuclease (TALEN; Figure 3—figure supplement 2).

DOI: http://dx.doi.org/10.7554/eLife.06120.008

Figure 3—figure supplement 1. CRISPR-mediated Fam20A KO and Fam20A/Fam20C double KO (DKO) in ALCs.

(A) The schematic of the sgRNA-targeting site in the mouse Fam20a gene and the disrupted allele in Fam20A-KO ALCs (clone A21). The first exon of the mouse Fam20a gene is shown as a box. White, 5′ UTR; Orange, the coding sequence. Targeting sites and PAMs are indicated with blue and red bars, respectively. Inserted nucleotides are highlighted in red. (B) Disrupted mouse Fam20c allele in Fam20A/Fam20C-DKO ALC (clone AC33). Fam20A-KO ALC clone A21 (Figure 3—figure supplement 1A) was used as the parental cell line. Fam20c gene was targeted using sgRNA m20c #3 (Figure 1—figure supplement 1A). (C) Endogenous Fam20C production by WT ALC or the KO clones used in this study. Cells were seeded at the same density and cultured overnight in serum-free media. Proteins in the conditioned media were precipitated using trichloroacetic acid (TCA) and separated by SDS-PAGE. Secreted Fam20C was detected by western blot (upper). Equal protein loading of each sample was shown by Ponceau S staining (lower).

DOI: http://dx.doi.org/10.7554/eLife.06120.009

Figure 3—figure supplement 2. Transcription activator-like effector nuclease (TALEN)-mediated Fam20C KO in U2OS cells.

(A) The schematic of the TALEN-targeting sites in the human FAM20C gene. (B) Disrupted human FAM20C allele in Fam20C-KO U2OS cells. Deleted region is indicated with red dashes. (C) Endogenous Fam20C production by WT or Fam20C-KO U2OS. The experiment was performed as in Figure 3—figure supplement 1C.

DOI: http://dx.doi.org/10.7554/eLife.06120.010