Journal: American Journal of Physiology - Renal Physiology

Article Title: Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome

doi: 10.1152/ajprenal.00207.2017

Figure Lengend Snippet: ADR-induced injury induced and activated small heat shock proteins (HSPs), primarily in an MK2-dependent manner. Total protein was extracted from renal cortexes isolated from control and ADR-injured mice on day 21 from each genotype and phosphorylated and total forms of HSPB1, HSPB8, glucose-regulated protein 78 (GRP78), and GAPDH were detected using their respective antibodies. A: representative Western blots of total and phosphorylated forms of selected proteins. B: densitometry analyses of protein induction/phosphorylation in the renal cortexes of at least 3 mice from each genotype. Statistical differences: *P < 0.05, control vs. ADR; #P < 0.05, WT-ADR treated vs. KO-ADR treated.

Article Snippet: The other primary antibodies used were anti-HSPB1 (StressMarq, Victoria, BC), anti-HSPB8 mouse monoclonal (Abcam, Cambridge, MA), anti-GRP78 rabbit monoclonal (StressMarq), and anti-GAPDH mouse monoclonal (Millipore, Billerica, MA).

Techniques: Isolation, Western Blot

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 2: The effect of stretch on EVs and endoplasmic reticulum (ER) stress in ASMCs. (A) Left to right: representative immunofluorescence images of CD63/HSPA5 in ASMCs acquired with confocal microscopy (×100 objective), the quantified CD63/HSPA5 puncta number inside ASMCs (ImageJ), and Pearson coefficient of the pixel-intensity correlation of CD63/HSPA5 in ASMCs (ImageJ, scale bar = 200 μm); (B) Protein expression of CD63/HSPA5 in ASMCs (WB); (C) Size distribution and quantified counts, as well as protein concentration of EVs in ASMCs (NTA and BCA). ER: endoplasmic reticulum; HSPA5: biomarker protein of ER stress; TUDCA: tauroursodeoxycholic acid, ER stress inhibitor; NTA, nanoparticle tracking analysis; BCA: bicinchoninic acid assay; data present as means ± SD, experiments were repeated three times (n = 3), **p < 0.01 compared with Static, ##p < 0.01 compared with Stretch

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Protein Concentration, Biomarker Discovery, Acid Assay

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 6: The effect of mechanical ventilation on secretion of EVs, ER stress, airway inflammation and injury in mouse models of VILI. (A) Representative images of lung tissue slides with hematoxylin and eosin (HE) or immunohisto- chemistry (IHC, HSPA5) staining (scale bar = 100 μm, arrows indicate alveolar collapse, arrowhead indicate HSPA5 expression). Healthy, MV, MV + TUDCA/GW4869 indicates mice breathed spontaneously, under 18 mL/kg mechanical ventilation (MV), MV with pretreatment of TUDCA/GW4869, respectively; (B) Protein expression of HSPA5 in lung tissue with IHC staining from C57BL/6 mice treated with different conditions, respectively; (C–E) Size distribution and quantified count, HSPA5, as well as protein concentration of EVs isolated from bronchoalveolar lavage fluid (BALF) of mice treated with different conditions; (F) Relative TGF-β1 and IL-10 secretion in BALF from mice treated with different conditions. Data present as means ± SD, experiments were repeated six times (n = 6), ** p < 0.01 compared with Healthy, #p < 0.05, ##p < 0.01 compared with MV

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunohistochemistry, Staining, Expressing, Protein Concentration, Isolation

Journal: Biology Open

Article Title: Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila

doi: 10.1242/bio.046524

Figure Lengend Snippet: Induction of UPR observed in wing imaginal discs and IPCs with ectopic Hsc70-3 DN expression. (A–B) Expression of Xbp1-GFP generated by ER stress-dependent splicing of xbp1*-GFP mRNA in wing imaginal discs. Phase contrast (A,B) and fluorescence (A′,B′) micrographs of wing imaginal discs. (A,A′) Control wing disc ( Bx>xbp1*-GFP ). (B,B′) Wing disc expressing a dominant-negative form of Hsc70-3 in the wing pouch region (arrow) ( Bx>hsc70-3 DN , xbp1*-GFP ). (C–E) Fluorescence micrograph of wing discs stained with DAPI (white). (C′–E′) Immunostaining of the wing discs with an anti-GRP78 antibody. (D″) Immunostaining of the wing disc with anti-HA antibody. (C,C′) Fluorescence micrograph of a control wing imaginal disc ( Bx-Gal4/+ ). (D–D″) Wing imaginal disc expressing control Hsc70-3 in the wing pouch region of the imaginal disc ( Bx>hsc70-3 ). (E,E′) Wing imaginal disc expressing a dominant-negative form of Hsc70-3 in the same region ( Bx>hsc70-3 DN ). Anti-GRP78 immunostaining is shown in white. Note that more intense immunofluorescence was observed exclusively in areas expressing Hsc70-3 DN , but not the control protein. (A–F) Relative intensity of anti-GRP78 immunostaining in wing imaginal discs. Immunofluorescence signal intensity in each wing imaginal disc with the control Hsc70-3 ( n =31) or Hsc70-3 DN ( n =25) expression was calculated and normalized to the control value, which was set as 1.0 ( Bx-Gal4/+ ) ( n =25; n.s., not significant, P >0.05; *** P <0.001, Student's t -tests). Error bars represent s.e.m. (G–I) Anti-GRP78 immunostaining of IPCs expressing GFPnls in brains from third-instar larvae. (G) Control IPCs ( ilp2>GFPnls ), (H) IPCs expressing the control Hsc70-3 ( ilp2>hsc70-3, GFPnls ), (I) IPCs expressing Hsc70-3 DN ( ilp2>hsc70-3 DN , GFPnls ). Anti-GRP78 immunostaining is colored in red (G–I; white in G′–I′). Nuclei of IPCs visualized by GFPnls expression are colored green (G–I; white in G″–I″). Arrows in H′ and H″ indicate positions of IPC cells. Note that remarkably higher immunostaining signal was observed in IPCs expressing Hsc70-3 DN , but not the control protein. (J) Relative intensities of anti-GRP78 immunostaining in larval IPCs. Immunofluorescence signal intensities in each IPC expressing Hsc70-3 ( n =25) or Hsc70-3 DN ( n =21) were calculated and normalized to the control value of 1.0 ( ilp2>GFPnls ) ( n =21, * P <0.05, *** P <0.001, Student's t -test). Error bars represent s.e.m. Scale bars: (A–E) 100 µm, (G–I) 50 µm.

Article Snippet: The following primary antibodies were used at the dilution described; rabbit anti-β-galactosidase (MP Biomedicals, #55976) at 1:1000, rabbit anti-GRP78 (Bip) (StressMarq Biosciences Inc., Cadboro Bay, Victoria, Canada) that could recognize Hsp70 family proteins including Hsc70-3 in Drosophila at 1:500, rabbit Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling, Danvers, Massachusetts, USA) at 1:200 for larval brain immunostaining and at 1:150 for wing disc immunostaining, rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) (Cell Signaling, antibody #9578) at 1:500, and rabbit anti-phospho-SAPK/JNK (pThr183, pTyr185) (Calbiochem, La Jolla, CA, USA) at 1:200.

Techniques: Expressing, Generated, Fluorescence, Dominant Negative Mutation, Staining, Immunostaining, Immunofluorescence

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