goat polyclonal anti human stc2  (R&D Systems)

Journal: Metabolism: clinical and experimental

Article Title: Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

doi: 10.1016/j.metabol.2022.155218

Figure Lengend Snippet: Fig. 1. PAPP-A is potentially regulated by STC1, STC2 and TGF-β in the mammalian kidney. Human kidney tissue was stained by immunohistochemistry using antibodies against PAPP-A (A), IGFBP-4 (B), STC1 (C), STC2 (D), or a control antibody (E). Scale bars represent 50 μm. (F), Antibodies against STC2 or STC1 were used for immunoprecipitation from human mesangial cell (HMC) culture medium or fresh serum medium as a control followed by PAPP-A Western blotting. Dimeric PAPP-A (400 kDa) forms a covalent 2:2 complex with STC2, which migrates with an apparent molecular weight of 500 kDa. On the contrary, the non-covalent PAPP-A:STC1 complex dissociates in denaturing SDS-PAGE, resulting in the appearance of the 400 kDa PAPP-A homodimer. Arrow and arrow head indicate the position of recombinant PAPP-A:STC2 complex (500 kDa) and recombinant PAPP-A dimer (400 kDa), respectively. The low molecular weight bands at approx. 150 kDa represent immunoglobulin used for IP. (G) mRNA was extracted from HMCs stimulated for 24 h with or without TGF-β, and mean PAPP-A expression ± SEM quantified by RT-PCR relative to RPL19 was calculated. n = 3 in each group. (H), mRNA was extracted from non-Tg or Tg-TGF-β mouse kidneys, and mean Papp-a expression ± SEM quantified by RT-PCR relative to G6pdh was calculated. n = 6 in each group. **p < 0.01.

Article Snippet: In experiments detecting proteins in culture supernatant, the membrane was incubated overnight at 4 ◦C with rabbit polyclonal anti-human PAPP-A [36], goat polyclonal anti-human STC2 (R&D Systems, AF2830), or biotinylated purified STC1, and then for 1 h at 22 ◦C with polyclonal swine anti-rabbit IgG-HRP (DAKO, P0217), polyclonal rabbit anti-goat IgG-HRP (DAKO, P0160), or avidinconjugated horseradish peroxidase (P0347, DAKO, Denmark).

Techniques: Staining, Immunohistochemistry, Control, Immunoprecipitation, Western Blot, Molecular Weight, SDS Page, Recombinant, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Metabolism: clinical and experimental

Article Title: Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

doi: 10.1016/j.metabol.2022.155218

Figure Lengend Snippet: Fig. 2. PAPP-A activity is increased in human dia betic kidney tissue relative to control. (A), Active PAPP-A is not inhibited and therefore accessible to STC1 in contrast to inhibited PAPP-A represented here by the PAPP-A:STC2 complex. By labelling STC1 with e.g., biotin, active PAPP-A, but not inhibited PAPP-A can be detected. (B), PAPP-A and PAPP-A:STC2 complex were probed with anti- PAPP-A antibody (left), anti-STC2 antibody (center) or biotinylated STC1 (right). The PAPP-A Western blot detects both active PAPP-A and PAPP-A in complex with STC2, the STC2 Western blot detects STC2 in complex with PAPP-A, and biotinylated STC1 detects free, active PAPP-A. Black arrow and arrow head indicate the position of recombinant PAPP-A: STC2 complex and recombinant PAPP-A, respec tively. (C), Representative images of sections of human kidney biopsies from diabetic patients or healthy donors subjected to immunofluorescence staining using DAPI (blue), a monoclonal antibody against PAPP-A (green) and biotinylated STC1 (red). White arrows indicate co-localization of total PAPP-A and active PAPP-A. Scale bars represent 50 μm. Glomerular total PAPP-A (D–E) or active PAPP-A (F–G) staining is shown. Intensity (D + F) or area of positive staining (E + G) above a set threshold of control and diabetic tissue were quantified and expressed relative to the control. (H), The glomerular active PAPP-A area in control (black circles) and diabetic (open circles) tissue is shown as a function of the glomerular total PAPP-A area. (I), The glomerular total PAPP-A- (black circles) and active PAPP-A (open circles)-positive area is depicted as a function of pa tient age at the time the kidney biopsy was taken. (J), The glomerular total PAPP-A (black circles) and active PAPP-A (open circles)-positive area is depicted as a function of eGFR at the time the kidney biopsy was taken. (D–J), Results are relative means ± SEM of n = 11 diabetic cases, and n = 9 controls. *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: In experiments detecting proteins in culture supernatant, the membrane was incubated overnight at 4 ◦C with rabbit polyclonal anti-human PAPP-A [36], goat polyclonal anti-human STC2 (R&D Systems, AF2830), or biotinylated purified STC1, and then for 1 h at 22 ◦C with polyclonal swine anti-rabbit IgG-HRP (DAKO, P0217), polyclonal rabbit anti-goat IgG-HRP (DAKO, P0160), or avidinconjugated horseradish peroxidase (P0347, DAKO, Denmark).

Techniques: Activity Assay, Control, Western Blot, Recombinant, Immunofluorescence, Staining

Journal: Metabolism: clinical and experimental

Article Title: Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

doi: 10.1016/j.metabol.2022.155218

Figure Lengend Snippet: Fig. 3. Treatment of diabetic mice with AAV-STC2 resulted in subtle differences in UAE and kidney/body weight ratio and attenuated diabetes-mediated glomerular hypertrophy. (A), AAV-derived STC2 and STC2(C120A) levels monitored throughout the study are shown. (B), Proteolytic activity against radiolabeled IGFBP-4 was assessed after incubation of recombinant murine PAPP-A with STC2 or STC2(C120A) immunoprecipitated from plasma of mice treated with saline, AAV-STC2, or AAV-C120A. PAPP-A-mediated cleavage of IGFBP-4 results in two IGFBP-4 fragments co-migrating in SDS-PAGE. Incubation of PAPP-A with AAV-derived STC2, but not STC2 (C120A), resulted in strongly reduced IGFBP-4 cleavage. Results are representative of three independent experiments. (C), Blood glucose (BG) levels monitored throughout the study are shown. The log transformed UAE (D), kidney/body weight ratio (E), and estimated mean glomerular volume (F) of saline-, AAV-STC2-, or AAV-C120A-treated non-diabetic (ND) or diabetic (D) mice are shown. In D, E and F, n = 5, 10, and 10, respectively, for ND saline; n = 10, 16, and 16 for D saline; n = 6, 8, and 7 for ND AAV-STC2; n = 11, 15, and 15 for D AAV-STC2; and n = 10, 11, and 17 for D AAV-C120A. Results are means ± SD *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns = not statistically significant.

Article Snippet: In experiments detecting proteins in culture supernatant, the membrane was incubated overnight at 4 ◦C with rabbit polyclonal anti-human PAPP-A [36], goat polyclonal anti-human STC2 (R&D Systems, AF2830), or biotinylated purified STC1, and then for 1 h at 22 ◦C with polyclonal swine anti-rabbit IgG-HRP (DAKO, P0217), polyclonal rabbit anti-goat IgG-HRP (DAKO, P0160), or avidinconjugated horseradish peroxidase (P0347, DAKO, Denmark).

Techniques: Derivative Assay, Activity Assay, Incubation, Recombinant, Immunoprecipitation, Clinical Proteomics, Saline, SDS Page, Transformation Assay

Journal: Metabolism: clinical and experimental

Article Title: Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

doi: 10.1016/j.metabol.2022.155218

Figure Lengend Snippet: Fig. 4. Hypothetical model of PAPP-A regulation in the mammalian kidney. In the normal kidney PAPP-A-mediated IGF signaling is regulated by STC1 or STC2, and an equilibrium between STC-inhibited PAPP-A (left) and active PAPP-A (right) exists. Prolonged hyperglycemia results in increased TGF-β expression, which shifts this equilibrium by upregulating PAPP-A, causing increased IGF signaling. Treatment with STC2 counteracts this shift by increasing the inhibitory activity in the kidney.

Article Snippet: In experiments detecting proteins in culture supernatant, the membrane was incubated overnight at 4 ◦C with rabbit polyclonal anti-human PAPP-A [36], goat polyclonal anti-human STC2 (R&D Systems, AF2830), or biotinylated purified STC1, and then for 1 h at 22 ◦C with polyclonal swine anti-rabbit IgG-HRP (DAKO, P0217), polyclonal rabbit anti-goat IgG-HRP (DAKO, P0160), or avidinconjugated horseradish peroxidase (P0347, DAKO, Denmark).

Techniques: Expressing, Activity Assay