1. Cell Biology
  2. Developmental Biology

SLC38A2 provides proline to fulfill unique synthetic demands arising during osteoblast differentiation and bone formation

  1. Leyao Shen
  2. Yilin Yu
  3. Yunji Zhou
  4. Shondra M Pruett-Miller
  5. Guo-Fang Zhang
  6. Courtney M Karner  Is a corresponding author
  1. Department of Orthopaedic Surgery, Duke University School of Medicine, United States
  2. Department of Internal Medicine, University of Texas Southwestern Medical Center, United States
  3. Department of Biostatistics and Bioinformatics, Duke University School of Medicine, United States
  4. Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, United States
  5. Sarah W. Stedman Nutrition and Metabolism Center & Duke Molecular Physiology Institute, Duke University Medical Center, United States
  6. Department of Medicine, Duke University School of Medicine, United States
  7. Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center at Dallas, United States
https://doi.org/10.7554/eLife.76963
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Cite this article
  1. Leyao Shen
  2. Yilin Yu
  3. Yunji Zhou
  4. Shondra M Pruett-Miller
  5. Guo-Fang Zhang
  6. Courtney M Karner
(2022)
SLC38A2 provides proline to fulfill unique synthetic demands arising during osteoblast differentiation and bone formation
eLife 11:e76963.
https://doi.org/10.7554/eLife.76963
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5 figures, 4 tables and 4 additional files

Figures

Figure 1 with 1 supplement
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Osteoblast proteins are enriched with the amino acid proline.

(A) Heat map depicting the relative amino acid enrichment for the indicated osteoblast proteins. Color bar represents fold enrichment relative to the average amino acid content. White boxes denote below-average enrichment. (B) Heat map depicting alanine or proline enrichment in differentiation-associated proteins. Color bar represents the percent increase in abundance relative to all proteins. (C) Volcano plot depicting the proline proportion of the top 500 genes that are induced or suppressed during osteoblast differentiation. Dashed lines denote quartiles while the solid line denotes the median. ****p≤0.00005 by unpaired two-tailed Student’s t-test. (D) Graphical depiction of the predicted change in demand for alanine or proline based on changes in gene expression during osteoblast differentiation. See numerical source data and RNAseq source data in Figure 1—source data 1.

Figure 1—source data 1

Numerical source data for Figure 1.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig1-data1-v3.zip
Figure 1—figure supplement 1
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Predicted amino acid demand changes during osteoblast differentiation.

(A) Functional assays of calvarial cells cultured in growth media (GM) or osteogenic medium (OM) for 10 days (n = 3). (B) qRT-PCR analysis of osteogenic marker genes Runx2, Sp7 (OSX), Col1a1, and Bglap in calvarial cells cultured in GM or OM for 7 days (n = 3). Error bars depict SD. (C) Graphical depiction of the predicted change in demand for cysteine based on changes in gene expression during osteoblast differentiation. See numerical source data in Figure 1—figure supplement 1—source data 1.

Figure 1—figure supplement 1—source data 1

Numerical source data for Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig1-figsupp1-data1-v3.zip
Figure 2 with 1 supplement
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Proline uptake and incorporation into protein increases during osteoblast differentiation.

(A) Graphical depiction of proline, glutamine, glutamate, aspartate, asparagine, serine, glycine, and alanine labeling from [U-13C]-proline (n = 3), [U-13C]-glutamine (n = 3), or [1,2-13C]-glucose (n = 3) in naïve calvarial osteoblasts. (B, C) Radiolabeled 3H-proline uptake assay performed in naive bone marrow stromal cells (BMSC) (n = 3) (B) or after 7 days of osteoblast differentiation (n = 3) (C). ****p≤0.00005 for osteogenic media (OM) vs. growth media (GM, denoted by dashed line), ####p≤0.00005 comparison between change in proline and glutamine by unpaired two-tailed Student’s t-test. (D) Radiolabeled proline incorporation assay performed in primary calvarial cells cultured in GM or OM for 7 days (n = 6). **p≤0.005 by unpaired two-tailed Student’s t-test. (E) Contribution of [U-13C]-proline or [U-13C]-glutamine to proline, glutamate, aspartate, alanine, or serine isolated from total protein (n = 3). See numerical source data and isotopomer-tracing source data in Figure 2—source data 1.

Figure 2—source data 1

Numerical source data for Figure 2.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig2-data1-v3.zip
Figure 2—figure supplement 1
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Proline uptake increases during osteoblast differentiation and does not contribute to TCA cycle intermediates.

(A, B) Radiolabeled proline or glutamine uptake assay performed in naïve calvarial osteoblasts (n = 3) (A) or after 7 days of osteoblast differentiation (n = 3) (B). Error bars depict SD. *p≤0.05, **p≤0.005 for growth media (GM) vs. osteogenic media (OM), ****p≤0.00005. #p≤0.05, ##p≤0.005 for proline vs. glutamine or alanine uptake, respectively, by unpaired two-tailed Student’s t-test. (C) Graphical depiction of aKG, malate, and citrate labeling from [U-13C]-proline (n = 3), [U-13C]-glutamine (n = 3), or [1,2-13C]-glucose (n = 3) after either 24 or 72 hr in naïve calvarial osteoblasts. See numerical source data in Figure 2—figure supplement 1—source data 1.

Figure 2—figure supplement 1—source data 1

Numerical source data for Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig2-figsupp1-data1-v3.zip
Figure 3 with 1 supplement
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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—source data 1

Numerical and uncropped western blot source data for Figure 3.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig3-data1-v3.zip
Figure 3—figure supplement 1
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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.

Figure 3—figure supplement 1—source data 1

Numerical and uncropped western blot source data for Figure 3—figure supplement 1.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig3-figsupp1-data1-v3.zip
Figure 4 with 1 supplement
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Slc38a2 provides proline critical for the synthesis of proline-rich proteins.

(A) Graphical depiction of the effects of 5 mM 2-(methylamino)-isobutyric acid (MeAIB) on radiolabeled amino acid uptake in primary calvarial cells (n = 3; except for glutamine n = 4). (B, C) Effect of Slc38a2 targeting on 3H-proline uptake (sgLuc n = 6; sgSlc38a2 n = 5) (B), or protein expression (n = 3) (C). 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. (D) Correlation analysis of protein expression as a function of the proline composition of proteins in Slc38a2 (sg38a2) targeted or control (sgLuc) calvarial cells. *p≤0.05, ***p≤0.0005, ****p≤0.00005 by unpaired two-tailed Student’s t-test. See numerical source data and uncropped Western blot images in Figure 4—source data 1.

Figure 4—source data 1

Numerical and uncropped western blot source data for Figure 4.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig4-data1-v3.zip
Figure 4—figure supplement 1
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Slc38a2 provides proline critical for the synthesis of proline-rich proteins.

(A) Graphical depiction of the effects of 5 mM 2-(methylamino)-isobutyric acid (MeAIB) on amino acid uptake in bones isolated from P3 mice (n = 3). (B) Schematic depicting Slc38a2 Crispr targeting strategy. (C–I) Effect of Slc38a2 targeting on intracellular amino acid concentration measured by mass spectrometry (n = 3) (C), contribution of [U-13C]-glutamine to proline (n = 3) (D), tRNA aminoacylation (n = 3) (E), protein expression (n = 3) (F), mRNA expression (n = 3) (G), protein (n = 3) (H), or collagen synthesis (n = 3) (I). In all blots, the percent proline composition is noted under the protein name. Protein expression normalized to total protein. Phosphorylated proteins are normalized to the total protein. Fold change ± SD for three independent experiments. *p≤0.05, ****p≤0.00005 by unpaired two-tailed Student’s t-test. See numerical source data and uncropped Western blot images in Figure 4—figure supplement 1—source data 1.

Figure 4—figure supplement 1—source data 1

Numerical and uncropped western blot source data for Figure 4—figure supplement 1.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig4-figsupp1-data1-v3.zip
Figure 5 with 3 supplements
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Slc38a2-dependent proline uptake is required for osteoblast differentiation during bone development.

(A–H) Skeletal preparations of Slc38a2LacZ/LacZ or wildtype controls (A, B, E, F) or Sp7Cre;Slc38a2fl/fl or Sp7Cre;Slc38a2fl/+ littermate controls (C, D, G, H) at embryonic day (E)15.5 (A–D) or P1 (E–H). Red arrow (A–D) or asterix (E–H) highlights reduced mineralization. A total of n = 7 or n = 5 Slc38a2LacZ/LacZ animals and n = 5 or n = 5 for Sp7Cre;Slc38a2fl/fl animals were analyzed at E15.5 or postnatal day (P)1, respectively. Scale bar = 5 mm. (I–R) Representative von Kossa staining (I, J), alkaline phosphatase (ALPL) staining (K, L) in situ hybridization for Spp1 (M, N), Ibsp (O, P), Bglap (Q, R), and Col1a1 (U, V), or immunofluorescent staining for OSX (S’, S”, T’, T”) and COL1A1 (W, X) on Sp7Cre;Slc38a2fl/fl (n = 4) (J, L, N, P, R, T, V, X) or Sp7Cre;Slc38a2fl/+ (n = 4) (I, K, M, O, Q, S, U, W) newborn calvariae. *p≤0.05 by paired two-tailed Student’s t-test. Scale bar = 100 μm.

Figure 5—figure supplement 1
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Slc38a2 is required for osteoblast differentiation in vitro.

(A–D) Effect of Slc38a2 targeting on cell viability (n = 3) (A), EdU incorporation (n = 3) (B), mRNA expression (n = 3) by qPCR analysis (C), or functional assays (D) in calvarial cells cultured in growth media (GM) or osteogenic medium (OM) for seven or 10 days (n = 3). Error bars depict SD.

*p≤0.05 by unpaired two-tailed Student’s t-test. See numerical source data in Figure 5—figure supplement 1—source data 1.

Figure 5—figure supplement 1—source data 1

Numerical source data for Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig5-figsupp1-data1-v3.zip
Figure 5—figure supplement 2
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Slc38a2LacZ/LacZ mutants have impaired osteoblast differentiation during endochondral ossification.

(A) Western blot analysis of SNAT2 expression in femur bone shaft protein isolated from Slc38a2LacZ/LacZ or wildtype controls. (B) Representative images of humerus skeletal preparations, von Kossa staining, alkaline phosphatase (ALPL) staining, in situ hybridization for Col1a1, or immunofluorescence staining for OSX and COL1A1 on femur sections of embryonic day (E)15.5 Slc38a2LacZ/LacZ or wildtype controls littermate controls (n = 3 animals). Scale bar = 200 μm. See uncropped Western blot images in Figure 5—figure supplement 2—source data 1.

Figure 5—figure supplement 2—source data 1

Uncropped western blot source data for Figure 5—figure supplement 2.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig5-figsupp2-data1-v3.zip
Figure 5—figure supplement 3
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Sp7Cre;Slc38a2fl/fl have impaired osteoblast differentiation during endochondral ossification.

(A) Western blot analysis of SNAT2 expression in femur bone shaft protein isolated from Sp7Cre;Slc38a2fl/fl or Sp7Cre;Slc38a2fl/+ littermate controls (n = 3). (B) Evaluation of amino acid uptake in femurs isolated from newborn Sp7Cre;Slc38a2fl/fl or Sp7Cre;Slc38a2fl/+ littermate controls (n = 5). (C) Skeletal preparations of newborn Sp7Cre;Slc38a2fl/fl or Sp7Cre;Slc38a2fl/+ littermate controls (n = 5). Scale bar in skeletal prep = 5 mm. Phalloidin staining or immunofluorescence staining for RUNX2 on postnatal day (P)0 Sp7Cre;Slc38a2fl/fl or Sp7Cre;Slc38a2fl/+ calvariae (n = 4). Scale bar = 100 μm. Boxed region shown in inset image. (D) Representative images of in situ hybridization for Spp1, Ibsp, Col1a1, and Sp7, or immunofluorescence staining for OSX, COL1A1, and COLX on humerus sections from embryonic day (E)15.5 Sp7Cre;Slc38a2fl/fl or Sp7Cre;Slc38a2fl/+ littermate controls (n = 4 animals). Red dashed line denotes the remodeling area. Fold change ± SD. Error bar depicts SD. *p≤0.05, ****p≤0.00005 by paired two-tailed Student’s t-test. Scale bar = 200 μm. See numerical source data and uncropped Western blot images in Figure 5—figure supplement 3—source data 1.

Figure 5—figure supplement 3—source data 1

Numerical and uncropped western blot source data for Figure 5—figure supplement 3.

https://cdn.elifesciences.org/articles/76963/elife-76963-fig5-figsupp3-data1-v3.zip

Tables

Table 1
Amino acid composition of classical osteoblast proteins.
RUNX2OSXCOL1A1OCNAll proteins
Ala0.0840.0820.0890.0740.068
Cys0.0120.0260.0120.0320.023
Asp0.0440.0330.0410.0630.048
Glu0.0210.0440.0520.0630.069
Phe0.0380.0230.0180.0210.038
Gly0.0530.1380.2680.0530.063
His0.0280.0440.0060.0000.026
Ile0.0210.0120.0170.0420.045
Lys0.0310.0510.0380.0630.057
Leu0.0590.0840.0350.1470.100
Met0.0230.0120.0100.0210.023
Asn0.0380.0260.0230.0320.036
Pro0.1050.1330.1900.0740.061
Gln0.0890.0330.0330.0320.048
Arg0.0560.0400.0470.0630.056
Ser0.1330.0980.0460.0740.085
Thr0.0690.0560.0300.0740.054
Val0.0540.0210.0290.0320.061
Trp0.0100.0140.0040.0000.012
Tyr0.0300.0300.0100.0420.027
Table 2
Relative amino acid composition of proteins associated with various differentiated cell types based on Gene Ontology (GO) terms.
OsteoblastEpithelial cellHematopoietic stem cell
Endothelial cellOsteoclastNeuronMuscle cellCardiomyocyteAll
GO term000016490030855003009700454460030316003018200426920055007
Ala0.07390.07310.06950.07090.07300.07280.07300.07310.0681
Cys0.03010.02300.02340.02590.02920.02280.02330.02230.0227
Asp0.04780.04830.04930.04670.04680.05020.05110.04900.0479
Glu0.06390.06780.06710.06820.06140.06930.07270.06900.0694
Phe0.03310.03310.03650.03420.03740.03470.03670.03730.0375
Gly0.06860.06980.06720.06850.06560.06670.06660.06670.0629
His0.02730.02560.02660.02390.02450.02540.02490.02560.0262
Ile0.03620.04010.04090.04580.04070.04200.04290.04410.0445
Lys0.05310.05570.05700.05310.04990.05600.06060.06270.0571
Leu0.09460.09230.09570.09500.10400.09610.09300.09030.1004
Met0.02200.02320.02290.02240.02100.02220.02220.02380.0228
Asn0.03600.03610.03620.03820.03660.03740.03690.03630.0360
Pro0.07110.06940.06590.06520.06500.06440.06270.06090.0612
Gln0.04690.04720.04680.04580.04460.04590.04640.04820.0478
Arg0.06070.05730.05630.05660.05390.05750.05610.05470.0559
Ser0.08530.08740.08480.08070.08430.08350.07970.08480.0853
Thr0.05270.05290.05370.05570.05630.05380.05280.05440.0543
Val0.05680.05880.05990.06390.06230.05980.05990.05850.0610
Trp0.01190.01100.01220.01190.01350.01170.01080.01050.0119
Tyr0.02780.02790.02800.02750.02980.02790.02750.02780.0270
Table 3
mRNA expression of putative proline transporters.
SystemAliascOB
FPKM
Slc38a2ASNAT28823.1
Slc1a4ASCASCT13030.7
Slc36a4LYAATPAT41929
Slc36a1LYAATPAT1803.7
Slc38a4ASNAT4267.9
Slc36a2LYAATPAT21.1
Slc6a15B0B0AT27.5
Slc36a3LYAATPAT30
Slc6a7IMINOBPROT4.3
Slc6a20aIMINOSIT20
Slc6a20bIMINOSIT10
Slc6a19B0B0AT10
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)C57Bl/6J Jackson Laboratory RRID:IMSR_JAX:000664
Genetic reagent (M. musculus)Rosa26Cas9 Jackson Laboratory RRID:IMSR_JAX:024858
Genetic reagent (M. musculus)Rosa26Flpe Jackson Laboratory RRID:IMSR_JAX:003946
Genetic reagent (M. musculus)Sp7tTA;tetOeGFP/Cre PMID:16854976 RRID:IMSR_JAX:006361
Genetic reagent (M. musculus)Slc38a2LacZ European Mouse Mutant ArchiveSee ‘Mouse strains’ for more information
Chemical compound, drugAscorbic acid Sigma Cat# A4544
Chemical compound, drugβ-Glycerophosphate Sigma Cat# G9422
Chemical compound, drugOne-step NBT/BCIP solution Thermo Fisher Cat# PI34042
Chemical compound, drugEasyTag EXPRESS S35 PerkinElmer Cat# NEG772002MC
Chemical compound, drugL-(3,4-3H)-Glutamine PerkinElmer Cat# NET551250UC
Chemical compound, drugL-[1,2-14C]-Alanine PerkinElmer Cat# NEC266E050UC
Chemical compound, drugL-(2,3-3H)-Alanine PerkinElmer Cat# NET348250UC
Chemical compound, drugL-(2,3,4-3H)-Proline PerkinElmer Cat# NET323250UC
Chemical compound, drugL-[3H(G)]-Serine PerkinElmer Cat# NET248250UC
Chemical compound, drugL-[14C(U)]-Glycine PerkinElmer Cat# NEC276E050UC
Chemical compound, drugL-[3,4-3H]-Glutamate PerkinElmer Cat# NET490001MC
Chemical compound, drugUltima Gold scintillation cocktail PerkinElmer Cat# 6013329
Chemical compound, drug[U-13C]-Glutamine Sigma Cat# 605166Used at 2 mM final concentration
Chemical compound, drug[U-13C]-Proline Cambridge Cat# 201740-83-2Used at 0.34 mM final concentration
Chemical compound, drug[1,2-13C]-Glucose Sigma Cat# 453188Used at 5.6 mM final concentration
Chemical compound, drugAP substrate BM purple Roche Cat# 11442074001
Chemical compound, drugECL substrate Bio-Rad Cat# 1705060
Chemical compound, drugSuper signal West Femto ECL Thermo Fisher Cat# 1705060
AntibodyEif2α (rabbit monoclonal) Cell Signaling RRID:AB_10692650(1:1000)
AntibodypSer51 Eif2α (rabbit monoclonal) Cell Signaling RRID:AB_2096481(1:1000)
AntibodypSer240/244 S6rp (rabbit polyclonal) Cell Signaling RRID:AB_331682(1:1000)
AntibodyS6rp (rabbit monoclonal) Cell Signaling RRID:AB_331355(1:1000)
Antibodyα-Tubulin (rabbit monoclonal) Cell Signaling RRID:AB_2619646(1:1000)
Antibodyβ-Actin (rabbit polyclonal) Cell Signaling RRID:AB_330288(1:1000)
AntibodyHRP goat anti-rabbit (goat polyclonal) Cell Signaling RRID:AB_2099233(1:2000)
AntibodyHRP anti-mouse (horse polyclonal) Cell Signaling RRID:AB_330924(1:2000)
AntibodyRunx2 (rabbit monoclonal) Cell Signaling RRID:AB_10949892(1:1000)
AntibodySmad1 (rabbit polyclonal) Cell Signaling RRID:AB_2107780(1:1000)
Antibody4E-BP1 (rabbit monoclonal) Cell Signaling RRID:AB_2097841(1:1000)
AntibodymTOR (rabbit monoclonal) Cell Signaling RRID:AB_2105622(1:1000)
AntibodyErk (rabbit monoclonal) Cell Signaling RRID:AB_390779(1:1000)
AntibodyeEF2 (rabbit polyclonal) Cell Signaling RRID:AB_10693546(1:1000)
AntibodyPhgdh (rabbit polyclonal) Cell Signaling RRID:AB_2750870(1:1000)
AntibodyAkt (rabbit polyclonal) Cell Signaling RRID:AB_329827(1:1000)
AntibodyCOL1A1 (mouse monoclonal) Santa Cruz RRID:AB_2797597(1:1000) WB(1:200) IF
AntibodyOSX (mouse monoclonal) Santa Cruz RRID:AB_2895257(1:1000) WB
AntibodyATF4 (rabbit polyclonal) Santa Cruz RRID:AB_2058752(1:1000)
AntibodyATF2 (mouse monoclonal) Santa Cruz RRID:AB_626708(1:1000)
AntibodyPAX1 (mouse monoclonal) Millipore Cat# MABE1115(1:1000)
AntibodySNAT2 (rabbit polyclonal) Abcam RRID:AB_2050321(1:1000)
AntibodyOSX (rabbit polyclonal) Abcam RRID:AB_2194492(1:200) IF
AntibodyCOLX (mouse monoclonal) Quartett Cat# 2031501217(1:200)
AntibodyGoat anti-mouse 568 (goat unknown clonality) Thermo Fisher RRID:AB_141359(1:200)
AntibodyGoat anti-rabbit 568 (goat polyclonal) Thermo Fisher RRID:AB_143157(1:200)
Commercial assay or kitAlexa Fluor 647 Phalloidin Thermo Fisher Cat# 22287
Commercial assay or kitIscript Reverse transcription kit Bio-Rad Cat# 1708841
Commercial assay or kitSYBR green Bio-Rad Cat# 1725275
Commercial assay or kitClick-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit Invitrogen Cat# C10420
Commercial assay or kitApoptosis Assay Kit (Cat# 22837) AAT BIO Cat# 22837
Chemical compound, drugAP substrate BM purple Roche Cat# 11442074001
Software, algorithmGraphPad 6 https://www.graphpad.com/ 
Software, algorithmR version 3.6.0 https://www.r-project.org/ 

Additional files

Supplementary file 1

sgRNA protospacer sequence.

https://cdn.elifesciences.org/articles/76963/elife-76963-supp1-v3.docx
Supplementary file 2

RT-PCR primer sequences for tRNA charging.

https://cdn.elifesciences.org/articles/76963/elife-76963-supp2-v3.docx
Supplementary file 3

RT-PCR primer sequences.

https://cdn.elifesciences.org/articles/76963/elife-76963-supp3-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/76963/elife-76963-transrepform1-v3.docx

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  1. Leyao Shen
  2. Yilin Yu
  3. Yunji Zhou
  4. Shondra M Pruett-Miller
  5. Guo-Fang Zhang
  6. Courtney M Karner
(2022)
SLC38A2 provides proline to fulfill unique synthetic demands arising during osteoblast differentiation and bone formation
eLife 11:e76963.
https://doi.org/10.7554/eLife.76963