Journal: BMC Complementary and Alternative Medicine

Article Title: Kihi-to, a herbal traditional medicine, improves Abeta(25–35)-induced memory impairment and losses of neurites and synapses

doi: 10.1186/1472-6882-8-49

Figure Lengend Snippet: Effects of Kihi-to on Aβ(25–35)-induced expression changes of calpain and calpastatin . Cortical neurons were cultured for 2 days and then treated with or without ( C ) 10 μM Aβ(25–35). The cells were simultaneously treated with Kihi-to (0.1 μg/ml, K ), MDL28170 (1 nM, M ), or vehicle ( V ) for 2, 8, 24 and 96 h, and then double-immunostained for calpain and MAP2 or for calpastatin and MAP2. Expression levels of calpain (A) and calpastatin (B) in MAP2-positive cells (neurons) were quantified. * p < 0.05 vs. Veh, n = 40. (one-way ANOVA followed by Holm-Sidak post hoc test).

Article Snippet: MDL28170 was purchased from Biomol (Plymouth Meeting, PA, USA).

Techniques: Expressing, Cell Culture

Journal: Science translational medicine

Article Title: Calpain 9 as a therapeutic target in TGFβ-induced mesenchymal transition and fibrosis *

doi: 10.1126/scitranslmed.aau2814

Figure Lengend Snippet: (A) Representative immunoblots (left, bracket indicates identical gel) and quantification (right, normalized to GAPDH) for the indicated proteins, time points after TGFβ stimulation, and concentrations of MDL-28170 (n =3 to 4). (B) Representative immunofluorescence images of TGFβ-induced EMT in NMuMG cells stained with E-cadherin (green), F-actin (red), and DAPI (blue). Scale bar: 100 µm. (C) Relative gene expression (normalized to Gapdh) in response to TGFβ with or without calpain inhibition (MDL) (n = 3). (D) Representative immunoblots (left) and quantification (right) for the indicated proteins, time points after TGFβ stimulation, and concentrations of calpeptin (n = 3). (E) Representative immunoblots (left) and quantification (right) for the indicated proteins, time points after TGFβ stimulation, and concentrations the calcium channel blocker 2-APB (n = 3). Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, †P < 0.005, ‡P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: MDL-28170 (Enzo Lifesciences, BML-PI130), calpeptin (Tocris, 0448), 2-APB (Sigma, D9754), CA-074-OMe (Sigma, C5857), SB431542 (Sigma, S4317), CCT128930 (Selleckchem, S2635), cycloheximide (Cell Signaling Technologies (CST), 2112S) were prepared in stock solutions according to the manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Endocannabinoids Prevent ?-Amyloid-mediated Lysosomal Destabilization in Cultured Neurons *

doi: 10.1074/jbc.M110.162040

Figure Lengend Snippet: Endocannabinoids prevent Aβ-induced calpain activation. A, Aβ significantly increased p-p53Ser-15 immunoreactivity (***, p < 0.001 versus control), and this was prevented by the calpain inhibitor, MDL 28170 (888, p < 0.001 versus Aβ). B, in cells exposed to Aβ, emission at 633 nm was significantly decreased (***, p < 0.001 versus control), and this was prevented by MDL 28170 (###, p < 0.001 versus Aβ). C, Aβ significantly increased DNA fragmentation in cultured cortical neurons (***, p < 0.001 versus control), and this was prevented by MDL 28170 (###, p < 0.001 versus Aβ). D, calpain activity was significantly increased in cultured cortical neurons exposed to Aβ (*, p < 0.05 versus control), and this was prevented by 2-AG, AEA, and URB 597 (#, p < 0.05 versus Aβ). n = 5. Error bars, S.E.

Article Snippet: The role of calpain in Aβ-induced toxicity was assessed using an inhibitor of its activity, MDL 28170 (10 μ m ) ( 6 ) (Merck).

Techniques: Activation Assay, Cell Culture, Activity Assay

Journal: Scientific Reports

Article Title: Cathepsins L and B target HIF1α for oxygen-independent proteolytic cleavage

doi: 10.1038/s41598-024-65537-9

Figure Lengend Snippet: HIF1αODD cleavage is reduced by cathepsins L or B inhibitors. ( a – c ). Rabbit reticulocyte lysate (RRL) was pre-incubated for 5 min at either 37 °C or 60 °C, with DMSO or inhibitor, as indicated. Recombinant HIF1αODD-GFP was added and incubated for 60 min at 37 °C. Reactions were resolved by SDS-PAGE and immunoblotted for GFP. E-64 was used at 10 μM; Q-VD-OPh, Z-VAD-FMK, calpastatin peptide, MDL-28170 calpeptin, CA-074 (CTSB inhibitor), LY3000328 (CTSS inhibitor), SB 412515 (CTSL inhibitor), and L-006,235 (CTSK inhibitor) were each used at 0.1, 1, and 10 μM. Blots are representative of three independent experiments. Original blots are presented in Supplementary Fig. .

Article Snippet: Z-VAD-FMK (Cat. # ALX-260-020-M001), Q-VD-OPh (Cat. # S7311), acetyl-calpastatin 184–210 (Cat. # 2950), MDL-28170 (Cat. # S7394), calpeptin (Cat. # 0448), LY3000328 (Cat. # 29729), SB 412515 (Cat. # 23249), L-006,235 (Cat. # 28843), recombinant human cathepsin B (Cat.# 28843), and recombinant human cathepsin L (Cat.# 787504) were obtained from Cedarlane Laboratories.

Techniques: Incubation, Recombinant, SDS Page