Figure 3. Proline is essential for the synthesis of proline-enriched osteoblast proteins.

(A) Western blot analyses of naïve calvarial cells cultured in 0.3 mM or 0 mM proline for 48 hr (n = 3). In all blots, the percent proline composition is noted under the protein name. Protein expression normalized to total protein. Fold change ± SD for three independent experiments. (B) Correlation analysis of protein expression as a function of the proline composition of proteins in naïve calvarial cells cultured in media containing either 0 mM or 0.3 mM proline for 48 hr. (C–G) The effect of proline availability on the synthesis of select proteins (n = 3). CHX, cycloheximide. Error bars depict SD. *p≤0.05 by unpaired two-tailed Student’s t-test. See numerical source data and uncropped Western blot images in Figure 3—source data 1.

Figure 3—figure supplement 1. Proline is essential for the synthesis of proline-enriched osteoblast proteins.

(A) Effect of 48 hr proline withdrawal on tRNA aminoacylation (n = 3). (B) Western blot analysis of naïve calvarial cells cultured in 0.3 mM or 0 mM proline for 48 hr (n = 3). Phosphorylated EIF2A normalized to total EIF2A while phosphorylated S6RP normalized to total S6RP. Individual proteins normalized to total protein. Fold change ± SD for three independent experiments. (C) qRT-PCR analysis of the effect of 48 hr proline withdrawal on gene expression in calvarial cells (n = 3). (D–F) The effect of proline availability on the synthesis of select proteins. CHX, cycloheximide. Fold change ± SD for three independent experiments. Error bars depict SD. *p≤0.05 by unpaired two-tailed Student’s t-test. See numerical source data and uncropped Western blot images in Figure 3—figure supplement 1—source data 1.