recombinant human catd  (Sino Biological)

Journal: Alzheimer's Research & Therapy

Article Title: Prominent tauopathy and intracellular β-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis

doi: 10.1186/s13195-024-01443-6

Figure Lengend Snippet: Inhibition of CatD slows the catabolism of tau in vitro and in cultured cells. ( A ) Representative Coomassie blue-stained polyacrylamide gel loaded with recombinant human tau (rTau) incubated for the indicated times with recombinant human CatD (5 nM) in the absence or presence of equal concentrations (1 µM) of Aβ40 or Aβ42. ( B ) Quantification of rTau levels as a function of time in 4 independent experiments. Note how rTau catabolism is unaffected by Aβ40 but markedly slowed by Aβ42, a potent competitive inhibitor of CatD. Data are mean ± SEM; n = 4. ( C ) Overview of the experimental approach used to quantify hTau catabolism in “Tet-Off” cultured neuroblastoma cells (see main text). ( D ) Representative western blot showing hTau levels (stained with antibody P44) at different time points after cessation of hTau expression in the absence or presence of the CatD inhibitor, pepstatin A (PepA; 1 µM). ( E ) Quantitation of hTau levels as a function of time from 6 independent experiments. Note the marked increase in the half-life of hTau in the presence of PepA (0.98 days; 95% CI 0.80 to 1.25) relative to DMSO-treated controls (0.51 days; 95% CI 0.429 to 0.627; P = 0.0012). Data are mean ± SEM, n = 6

Article Snippet: Reactions were initiated by addition of purified human CatD (2.5 nM; Enzo Life Sciences, Farmingdale, NY) dissolved in Assay Buffer, then 20-µL aliquots were removed 0, 0.5, 1, 2, and 4 h thereafter, with CatD activity in each aliquot immediately terminated by addition of PepA (1 µM) and incubation on ice.

Techniques: Inhibition, In Vitro, Cell Culture, Staining, Recombinant, Incubation, Western Blot, Expressing, Quantitation Assay

Journal: Cells

Article Title: Palmitic and Stearic Acids Inhibit Chaperone-Mediated Autophagy (CMA) in POMC-like Neurons In Vitro

doi: 10.3390/cells11060920

Figure Lengend Snippet: Lysosomal LAMP2A protein levels decrease in POMC-like neurons treated with PA and SA. ( A ) POMC-like neurons (N43/5) were incubated with 100 mM of PA, SA, or vehicle (BSA) for 6 h and a mitochondrial and lysosomal fractionation was carried out according to Ormeño et al. 2020. Post nuclear (PN), lysosomal (LYS), and mitochondrial (MIT) fractions were evaluated by Western blot using LAMP1, CATD, LAMP2, VDAC, and MTCO1 antibodies, as appropriate. ( B ) Representative Western blot comparing the lysosomal LAMP2A protein levels versus the PN fractions after BSA, PA, and SA treatment. ( C ) Quantification of LAMP2A from B using ordinary one-way ANOVA ( n = 3). n.s. = non-significant, ** = p < 0.01.

Article Snippet: The supernatant was discarded and pellets (10–20 μg) were used to evaluate the fractionation by Western blot using the antibodies LAMP2A (Abcam #ab18528, Cambridge, UK), LAMP1 (Cell Signaling #9091S, Beverly, MA, USA), CATD (R&D Systems #AF1014, Minneapolis, MN, USA), VDAC (Cell Signaling #4866S.

Techniques: Incubation, Fractionation, Western Blot

Journal: eLife

Article Title: KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

doi: 10.7554/eLife.44478

Figure Lengend Snippet: ( A ) Cleavage of VEGF-C by coincubation of pro-VEGF-C with cathepsin D (left panel) and secondary activation of ΔNΔC-VEGF-C (a mature form of VEGF-C translated from a truncated cDNA, right panel). ( B ) Quantification of the cleavage of pro-VEGF-C (n = 3) and ΔNΔC-VEGF-C (n = 2) by cathepsin D. ( C ) Cathepsin-D-mediated conversion of pro-VEGF-D into mature VEGF-D (left panel), and rapid activation of ΔNΔC-VEGF-D (a mature form of VEGF-D translated from a truncated cDNA, right panel). ( D ) Cleavage of pro-VEGF-D by KLK3. Note that, KLK3 cleaves VEGF-D between the VEGF homology domain and the N-terminal propeptide, but also between the VEGF homology domain and the C-terminal propeptide (for a detailed breakdown of the cleavage products visible in this overlay and the individual exposures, see ) (n = 2). 10.7554/eLife.44478.021 Figure 6—source data 1. Quantification of the cleavage of pro-VEGF-C and mature VEGF-C by cathepsin D.

Article Snippet: Recombinant protein , Cathepsin D (CATD) , R and D Systems/ Biotechne , 1014-AS , .

Techniques: Activation Assay

Journal: eLife

Article Title: KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

doi: 10.7554/eLife.44478

Figure Lengend Snippet: Shown are the results of Ba/F3-VEGFR/EpoR assays used to evaluate the receptor-activating properties of cathepsin D- and KLK3- cleaved proteins. Cathepsin D-cleaved VEGF-C activity in the ( A ) Ba/F3-VEGFR-3/EpoR assay and ( B ) Ba/F3-VEGFR-2/EpoR assay. ( C ) Mature VEGF-D after secondary activation with cathepsin D in the Ba/F3-VEGFR-3/EpoR assay. ( D ) The minor form of mature VEGF-D generated by cathepsin D-cleavage is less active than the major mature form in the Ba/F3-VEGFR-2/EpoR assay. ( E ) KLK3 activation of VEGF-D increases its potency in the Ba/F3-VEGFR-2/EpoR assay. ( F ) The secondary activation of mature VEGF-C with cathepsin D led to a small decrease in the response of Ba/F3-VEGFR-2/EpoR cells (n = 2). Error bars indicate SD. 10.7554/eLife.44478.025 Figure 7—source data 1. Ba/F3 assay showing the receptor-activating properties of cathepsin D-cleaved VEGF-C and VEGF-D.

Article Snippet: Recombinant protein , Cathepsin D (CATD) , R and D Systems/ Biotechne , 1014-AS , .

Techniques: Activity Assay, Activation Assay, Generated

Journal: eLife

Article Title: KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

doi: 10.7554/eLife.44478

Figure Lengend Snippet: Pro-VEGF-D cleaved by cathepsin D stimulated the phosphorylation of VEGFR-2 expressed in PAE cells at 40 ng/ml and 80 ng/ml concentrations.

Article Snippet: Recombinant protein , Cathepsin D (CATD) , R and D Systems/ Biotechne , 1014-AS , .

Techniques:

Journal: eLife

Article Title: KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

doi: 10.7554/eLife.44478

Figure Lengend Snippet: ( A ) Shown are the immunofluorescent stainings of the blood and lymphatic vessels in skeletal muscle transduced with recombinant adeno-associated virus subtype 9 (AAV9) encoding the KLK3- or the cathepsin D (CATD)-form of VEGF-C. Quantification of ( B ) Lyve-1 positive stained area and ( C ) CD31 positive stained area in AAV9 transduced tibialis anterior muscle. Data are presented as mean ± SEM, n = 4, one-way ANOVA with Tukey’s post hoc test, ***p<0.001, *p<0.05. Scale bar, 100 µm. (see also ). 10.7554/eLife.44478.029 Figure 8—source data 1. In vivo quantification of lymphangiogenesis and angiogenesis induced by KLK3- and cathepsin D-forms of VEGF-C.

Article Snippet: Recombinant protein , Cathepsin D (CATD) , R and D Systems/ Biotechne , 1014-AS , .

Techniques: Transduction, Recombinant, Virus, Staining, In Vivo

Journal: eLife

Article Title: KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

doi: 10.7554/eLife.44478

Figure Lengend Snippet: Comparison of VEGF-C mRNA levels in TA muscle transduced with AAV9 encoding for the different forms of mature VEGF-C generated by ADAMTS3 (Pos Ctrl), cathepsin D (CATD-form) and KLK3 (KLK3-form). Data are presented as mean ± SEM.

Article Snippet: Recombinant protein , Cathepsin D (CATD) , R and D Systems/ Biotechne , 1014-AS , .

Techniques: Comparison, Transduction, Generated

Journal: eLife

Article Title: KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

doi: 10.7554/eLife.44478

Figure Lengend Snippet:

Article Snippet: Recombinant protein , Cathepsin D (CATD) , R and D Systems/ Biotechne , 1014-AS , .

Techniques: Expressing, Transfection, Construct, Mutagenesis, Recombinant, Virus, Positive Control, Negative Control, Transformation Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Plasmid Preparation