![]() Error bars present the mean ± standard deviation (* p < 0.05). Student t-test was used for statistical analysis. (B) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Fl-APP and BACE1 were monitored in cell lysate, APP-CTFs were immunoprecipitated form the cell lysate. (A) Western blot of CHOwt cells treated with CtsB/L (PADK) and CtsD (PepA) inhibitors. Error bars present the mean ± standard deviation (** p < 0.01).Ĭathepsin B/L are essential in lysosomal degradation of the key Alzheimer’s proteins. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. (B) Western blot analysis of CtsD KO MEFs using LC3, LAMP1 and NPC1 antibody. Cholesterol (filipin staining, white) and NPC1 (green). Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).Ĭathepsin D genetic depletion in MEF cells does not cause cholesterol nor lysosomal proteins accumulation. ![]() (B) Western blot analysis of CtsB KO, CtsL KO and CtsB/L double KO MEFs using LC3, LAMP1 and NPC1 antibody. (A) Confocal microscopy of CtsB KO, CtsL KO and CtsB/L double KO MEFs. The error bars present the mean ± SEM (* p < 0.05).Ĭathepsin B/L genetic depletion in MEF cells causes accumulation of cholesterol and lysosomal proteins. ATP-Binding Cassette sub-family A member 1 (ABCA1) and Niemann-Pick C1 protein (NPC1) mRNA levels presented as a fold change. (D) RT-PCR Expression of the cholesterol egress genes in the control, PADK, U18666A- and Leu/NH 4Cl-treated SH-SY5Y cells, normalized to β-actin and quantified by 2−ΔΔCt method using control sample as calibrator. Error bars present the mean ± standard deviation (*** p < 0.001). (B) Western blot of SH-SY5Y control and PADK treated cells using LC3, ABCA1 and NPC1 antibody. (A) Confocal microscopy of SH-SY5Y control and PADK treated cells. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).Ĭathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in SH-SY5Y cells. (B) Western blot of CHOwt cells treated with different inhibitors using ABCA1, NPC1 and NPC2 antibody. (A) Confocal microscopy of CHOwt cells treated with different inhibitors. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01).Ĭathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in CHO cells. (E) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. (D) Western blot of CHOwt cells treated with different inhibitors and CHO NPC1-null cells using LC3 antibody. (C) Confocal microscopy of CHOwt cells treated with different inhibitors and CHO NPC1-null cells. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01). ![]() Western blot analysis of EGFR in SH-SY5Y cells treated with different inhibitors at different time points. Inhibition of cathepsin B/L and not cathepsin D causes lysosomal dysfunction.
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