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.