Human Pappalysin-1/PAPP-A Antibody

Detection of Human Pappalysin‑1/ PAPP‑A by Western Blot. Western blot shows lysate of human pregnant sera. PVDF membrane was probed with 0.1 µg/mL of Goat Anti-Human Pappalysin-1/PAPP-A Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2487) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Pappalysin-1/PAPP-A at approximately 200 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Pappalysin-1/PAPP-A by Western Blot PAPPA KO-EVs were generated using CRISPR/Cas9.a Schematic depicting hypothesized mechanism of (intra)cellular signalling activated by EV-associated PAPP-A, based on identified proteins and significantly altered phosphosites measured in HMEC-1 upon veh-EV stimulation by (phopho)proteomic analysis. Detected proteins in HMEC-1 are displayed in grey, while significantly changing phosphosites present in cluster C1 (see Fig. 3d) are displayed in brown. b Representative western blot analysis of phosphorylated AKT (pAKT), total AKT (tAKT), phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) in HMEC-1 treated with 6 × 1010 or 2 × 1010 CPC-EVs, or with 200 ng/mL free IGF-1 after pre-incubation with different doses of picropodophyllin (PPP). beta -actin ( beta -ACT) was included as housekeeping protein (I = phosphorylated protein blot, II = total protein blot). Biological replicates of (b) are displayed in Supplementary Fig. 9g, h. c Sanger sequencing results confirming 1 bp insertion in exon 3 of PAPPA at the CRISPR/Cas9 target site of the PAPPA KO-CPC clone, compared with the NTgRNA polyclonal CPC line. d Western blot analysis showing the absence of PAPP-A in PAPPA KO-EVs, compared with NTgRNA-EVs; the presence of CD81, CD63, Syntenin-1 (SYNT), Flotillin (FLOT1), beta -ACT, and absence of Calnexin (CNX) in both EV populations. FLOT1, beta -ACT and CNX were present in CPC lysate (CL). e Representative NTA plot showing the size distribution and particle concentration of PAPPA KO- and NTgRNA-CPC-EVs. f Protein content per 1 × 1010 PAPPA KO- and NTgRNA-EVs of two representative experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37528162), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Pappalysin-1/PAPP-A by Western Blot PAPPA KO-EVs were generated using CRISPR/Cas9.a Schematic depicting hypothesized mechanism of (intra)cellular signalling activated by EV-associated PAPP-A, based on identified proteins and significantly altered phosphosites measured in HMEC-1 upon veh-EV stimulation by (phopho)proteomic analysis. Detected proteins in HMEC-1 are displayed in grey, while significantly changing phosphosites present in cluster C1 (see Fig. 3d) are displayed in brown. b Representative western blot analysis of phosphorylated AKT (pAKT), total AKT (tAKT), phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) in HMEC-1 treated with 6 × 1010 or 2 × 1010 CPC-EVs, or with 200 ng/mL free IGF-1 after pre-incubation with different doses of picropodophyllin (PPP). beta -actin ( beta -ACT) was included as housekeeping protein (I = phosphorylated protein blot, II = total protein blot). Biological replicates of (b) are displayed in Supplementary Fig. 9g, h. c Sanger sequencing results confirming 1 bp insertion in exon 3 of PAPPA at the CRISPR/Cas9 target site of the PAPPA KO-CPC clone, compared with the NTgRNA polyclonal CPC line. d Western blot analysis showing the absence of PAPP-A in PAPPA KO-EVs, compared with NTgRNA-EVs; the presence of CD81, CD63, Syntenin-1 (SYNT), Flotillin (FLOT1), beta -ACT, and absence of Calnexin (CNX) in both EV populations. FLOT1, beta -ACT and CNX were present in CPC lysate (CL). e Representative NTA plot showing the size distribution and particle concentration of PAPPA KO- and NTgRNA-CPC-EVs. f Protein content per 1 × 1010 PAPPA KO- and NTgRNA-EVs of two representative experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37528162), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Pappalysin-1/PAPP-A by Western Blot LC–MS/MS identified enriched proteins in veh-EVs compared with Ca ion- and SKOV-3-EVs.a Heat map of protein abundance (log2) of proteins identified in each biological replicate (veh-, Ca ion- and SKOV-3-EVs), as identified by LC–MS/MS. b Volcano plots showing average fold changes for protein abundance (log2) of proteins identified in veh-EVs compared to (i) Ca ion-EVs and (ii) SKOV-3-EVs. P-values were calculated using student’s T-test, and significantly changing proteins (p-value ≤ 0.05 and fold change >2) in veh-EVs are highlighted in red, while significantly changing proteins in Ca ion- and SKOV-3-EVs are highlighted in blue. c Western blot analysis confirming the enrichment of MS-identified proteins NID1, TSG-6, LAMC1, PAPP-A, CD81 and beta -actin ( beta -ACT) in veh-EVs compared with Ca ion-EVs. CNX was solely present in CPC lysate (CL). Complete blots of beta -ACT, PAPP-A and NID1 are displayed in Supplementary Fig. 10. d Venn diagram showing number of proteins with >2-fold significant enrichment (p ≤ 0.05) in veh-EVs compared to Ca ion- and SKOV-3-EVs, and overlap between those two populations. e Gene ontology analysis using PANTHER of enriched biological processes for the 105 overlapping proteins, depicting number of identified proteins in each group, ranked on smallest corrected p-value (−log10(FDR)). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37528162), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Pappalysin-1/PAPP-A by Western Blot PAPPA KO-EVs showed reduced activation of intracellular signalling and activation of HMEC-1 migration and sprout formation.a–d Representative western blot analyses of pAKT, tAKT, pERK1/2 and tERK1/2 in HMEC-1 treated with PAPPA KO- and NTgRNA-EVs normalized on two doses of a, b total particle numbers or c, d total protein content. beta -ACT was included as housekeeping protein. b, d Quantification of pAKT, tAKT, pERK1/2 and tERK1/2 expression levels using densitometry expressed as pAKT/AKT and pERK/ERK ratios (n = 3). Biological replicates of (a, c) are also displayed in Supplementary Fig. 9i–k. e Wound healing assay showing effects of 1 µg and 2 × 1010 NTgRNA- and PAPPA KO-EVs on HMEC-1 migration, analysed both as % wound closure and absolute migration distance (n = 3, technical replicates. Data are representative of three biologically independent experiments). f, g Sprouting assay showing NTgRNA- and PAPPA KO-EV-induced HMEC-1 sprout formation on beads, analysed both as (g) mean length per sprout and total sprout length per bead (n = 3, technical replicates. Data are representative of two biologically independent experiments). Data are presented as mean ± SD. *p < 0.033, **p < 0.0021, ***p < 0.0002. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37528162), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Pappalysin-1/PAPP-A by Western Blot LC–MS/MS identified enriched proteins in veh-EVs compared with Ca ion- and SKOV-3-EVs.a Heat map of protein abundance (log2) of proteins identified in each biological replicate (veh-, Ca ion- and SKOV-3-EVs), as identified by LC–MS/MS. b Volcano plots showing average fold changes for protein abundance (log2) of proteins identified in veh-EVs compared to (i) Ca ion-EVs and (ii) SKOV-3-EVs. P-values were calculated using student’s T-test, and significantly changing proteins (p-value ≤ 0.05 and fold change >2) in veh-EVs are highlighted in red, while significantly changing proteins in Ca ion- and SKOV-3-EVs are highlighted in blue. c Western blot analysis confirming the enrichment of MS-identified proteins NID1, TSG-6, LAMC1, PAPP-A, CD81 and beta -actin ( beta -ACT) in veh-EVs compared with Ca ion-EVs. CNX was solely present in CPC lysate (CL). Complete blots of beta -ACT, PAPP-A and NID1 are displayed in Supplementary Fig. 10. d Venn diagram showing number of proteins with >2-fold significant enrichment (p ≤ 0.05) in veh-EVs compared to Ca ion- and SKOV-3-EVs, and overlap between those two populations. e Gene ontology analysis using PANTHER of enriched biological processes for the 105 overlapping proteins, depicting number of identified proteins in each group, ranked on smallest corrected p-value (−log10(FDR)). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37528162), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Pappalysin-1/PAPP-A by Western Blot PAPPA KO-EVs showed reduced activation of intracellular signalling and activation of HMEC-1 migration and sprout formation.a–d Representative western blot analyses of pAKT, tAKT, pERK1/2 and tERK1/2 in HMEC-1 treated with PAPPA KO- and NTgRNA-EVs normalized on two doses of a, b total particle numbers or c, d total protein content. beta -ACT was included as housekeeping protein. b, d Quantification of pAKT, tAKT, pERK1/2 and tERK1/2 expression levels using densitometry expressed as pAKT/AKT and pERK/ERK ratios (n = 3). Biological replicates of (a, c) are also displayed in Supplementary Fig. 9i–k. e Wound healing assay showing effects of 1 µg and 2 × 1010 NTgRNA- and PAPPA KO-EVs on HMEC-1 migration, analysed both as % wound closure and absolute migration distance (n = 3, technical replicates. Data are representative of three biologically independent experiments). f, g Sprouting assay showing NTgRNA- and PAPPA KO-EV-induced HMEC-1 sprout formation on beads, analysed both as (g) mean length per sprout and total sprout length per bead (n = 3, technical replicates. Data are representative of two biologically independent experiments). Data are presented as mean ± SD. *p < 0.033, **p < 0.0021, ***p < 0.0002. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37528162), licensed under a CC-BY license. Not internally tested by R&D Systems.