Journal: Pediatric research

Article Title: Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth.

doi: 10.1038/s41390-023-02501-9

Figure Lengend Snippet: Fig. 1 Flow chart of the present study. We previously used proteomics37 to identify six proteins, including HSPA5, with expression levels associated with SPTB. We then used WES to identify potentially damaging variants in families with recurrent SPTBs.13,37 In this study, we validated the proteomic result, and investigated the function of HSPA5 in SPTB by immunohistochem- istry, immunoelectron microscopy, and siRNA-mediated gene silencing.

Article Snippet: We used mouse monoclonal antihuman HSPA5 antibody (MAB4846, 1:1000 dilution; R&D Systems, Minneapolis, Minnesota) and rabbit monoclonal anti-human tubulin α-1B antibody (NB110-57609, 1:5000 dilution; Novusbio, Abingdon, United Kingdom) to detect HSPA5 and tubulin α-1B, respectively (Supplementary Fig. S1).

Techniques: Expressing, Immuno-Electron Microscopy

Journal: Pediatric research

Article Title: Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth.

doi: 10.1038/s41390-023-02501-9

Figure Lengend Snippet: Fig. 3 Crystal structure of human HSPA5. HSPA5 has a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD).42,44 a Crystal structure of HSPA5 when ATP is bound in the NBD (PDB code 5E84.42) SBD is in open conformation. b Crystal structure of HSPA5 with SBD in closed conformation (PDB code 5E85.42) This crystal structure lacks the NBD. C-terminal alpha helices, also known as the lid, cover the substrate in the binding pocket.25,42,44 Substrate (peptide substrate for DnaK [NR peptide]) in SBD is shown in stick representation, where oxygen, nitrogen, and carbon are colored in red, blue, and green, respectively. c Superposition of open and closed conformations shown in (a) and (b) using SBD only. Structures (a–c) are drawn as ribbons, and the site of the amino acid change (E557G) is displayed as a stick drawing. Open and the closed conformations are colored in pink and cyan, respectively. Crystal structures were illustrated with PyMOL.

Article Snippet: We used mouse monoclonal antihuman HSPA5 antibody (MAB4846, 1:1000 dilution; R&D Systems, Minneapolis, Minnesota) and rabbit monoclonal anti-human tubulin α-1B antibody (NB110-57609, 1:5000 dilution; Novusbio, Abingdon, United Kingdom) to detect HSPA5 and tubulin α-1B, respectively (Supplementary Fig. S1).

Techniques: Binding Assay

Journal: Pediatric research

Article Title: Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth.

doi: 10.1038/s41390-023-02501-9

Figure Lengend Snippet: Fig. 2 Placental protein expression of HSPA and mRNA expression of HSPA5 in SPTBs and STBs. Placental samples were from the basal plate of the placenta. Protein expression was used to validate the proteomic finding of HSPA5 as described previously.37 Protein levels normalized against the reference protein tubulin α-1B (a). Relative mRNA expression of HSPA5 assessed by qPCR. mRNA levels normalized against the housekeeping gene CYC1 (b). Statistical analysis was performed with Mann–Whitney U test to discover differences. Expression ratio (fold change [FC]) between compared groups presented in the figures. Quartiles displayed by a box and whiskers. Ends of whiskers represent minimum and maximum values, excluding outliers. Inside box, median is indicated with a line and mean value is represented as a square.

Article Snippet: We used mouse monoclonal antihuman HSPA5 antibody (MAB4846, 1:1000 dilution; R&D Systems, Minneapolis, Minnesota) and rabbit monoclonal anti-human tubulin α-1B antibody (NB110-57609, 1:5000 dilution; Novusbio, Abingdon, United Kingdom) to detect HSPA5 and tubulin α-1B, respectively (Supplementary Fig. S1).

Techniques: Expressing, MANN-WHITNEY

Journal: Pediatric research

Article Title: Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth.

doi: 10.1038/s41390-023-02501-9

Figure Lengend Snippet: Fig. 4 Placental localization of HSPA5 in spontaneous preterm and term births. Localization of HSPA5 in SPTB and STB placentas. In total, 12 placentas (SPTBs n = 6, STBs n = 6) were immunostained with anti‐human HSPA5 antibody. Samples were from the basal plate (maternal side) of the placenta. Immunostaining is indicated by filled large arrows in cytotrophoblast, unfilled large arrows in syncytiotrophoblast, filled small arrows in decidual trophoblast, and filled arrowheads in capillary endothelial cells. Original magnifica- tion is ×20 in all figures. Control represents the isotype controls for immunostaining. Scale bar represents 100 μm.

Article Snippet: We used mouse monoclonal antihuman HSPA5 antibody (MAB4846, 1:1000 dilution; R&D Systems, Minneapolis, Minnesota) and rabbit monoclonal anti-human tubulin α-1B antibody (NB110-57609, 1:5000 dilution; Novusbio, Abingdon, United Kingdom) to detect HSPA5 and tubulin α-1B, respectively (Supplementary Fig. S1).

Techniques: Immunostaining, Control