Extended Data Fig. 1: PPIA mass spectrometry and limiting dilution assay. | Nature Cell Biology

Extended Data Fig. 1: PPIA mass spectrometry and limiting dilution assay.

From: Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

Extended Data Fig. 1

a, Identification coverage of PPIA. Spots were excised from 2-D SDS PAGE for extraction and trypsin digestion. MS/MS spectra cover 49.8% of the entire PPIA protein sequence (underlined; representative of two independent mass spectrometry identifications). b, Analysis of proteomic data in the haematopoietic compartment based on Zaro et al.16. PPIA is robustly expressed in haematopoietic stem cells (HSCs), but also expressed in progenitor compartments throughout haematopoiesis. Violin plots represent PPIA abundance in six replicate animals. MPP, multipotent progenitors; CLP, common lymphoid progenitors; CMP, common myeloid progenitors; MEP, megakaryocyte-erythroid progenitors; GMP, granulocyte-monocyte progenitors. c, Phylogenetic tree of the Cyclophilin protein family in humans. The tree is based on protein sequence alignments from the NCBI RefSeq database with Clustal Omega. The red box indicates PPIA. Accession numbers for the individual proteins are: PPIL6 (NP_775943.1), NKTR (NP_005376.2), PPIG (NP_004783.2), PPIE (NP_006103.1) PPIH (NP_006338.1), PPID (NP_005029.1), RANBP2 (NP_006258.3), PPIA (NP_066953.1), PPIF (NP_005720.1), PPIB (NP_000933.1), PPIC (NP_000934.1), SDCCAG-10 (NP_005860.2), PPIL1 (NP_057143.1), PPIL2 (NP_055152.1), PPIL3 (NP_115861.1), PPIL4 (NP_624311.1), and PPWD1 (NP_056157.1). d, Limiting dilution transplantations of Ppia heterozygous (Ppia+/−) and knockout (Ppia−/−) bone marrow. 500,000 competitor cells (CD45.1+) were co-injected with 4,000, 20,000, 100,000, or 500,000 nucleated bone marrow cells of Ppia+/− or Ppia−/− mice. Reconstitution of peripheral CD45.2+ cells was assayed 20 weeks after transplantation and differences were compared using a two-tailed Poisson t-test. No significant difference exists between Ppia heterozygous and knockout donors; (a representative example of two independent experiments is shown with n = 5 animals per group for the two high donor cell doses and n = 10 animals per group for the two low donor cell doses).

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