Inhibition of cathepsin B/L and not cathepsin D causes lysosomal dysfunction. Western blot analysis of EGFR in SH-SY5Y cells treated with different inhibitors at different time points. (B) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01). (C) Confocal microscopy of CHOwt cells treated with different inhibitors and CHO NPC1-null cells. (D) Western blot of CHOwt cells treated with different inhibitors and CHO NPC1-null cells using LC3 antibody. (E) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. 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. (A) Confocal microscopy of CHOwt cells treated with different inhibitors. (B) Western blot of CHOwt cells treated with different inhibitors using ABCA1, NPC1 and NPC2 antibody. 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. (A) Confocal microscopy of SH-SY5Y control and PADK treated cells. Cholesterol (filipin staining, white) and NPC1 (green). However, to get meaningful results quantification requires careful consideration of background, saturation, normalization, and statistical analysis. (B) Western blot of SH-SY5Y control and PADK treated cells using LC3, ABCA1 and NPC1 antibody. Western blot quantification can give you the ability to study changes in protein levels. 1) Open western image in Image J, select Rectangular Selections tool from the ImageJ toolbar and select first western band. Although the Lite version is free, there is a more comprehensive paid version of the software that aims to easily. Image Studio Lite is a free software package from LI-COR Biosciences aimed at life scientists that want to analyze gels, western blots, dot blots, and other similar lab outputs. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Analyzing western blots with Image Studio Lite. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (*** p < 0.001). (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. ATP-Binding Cassette sub-family A member 1 (ABCA1) and Niemann-Pick C1 protein (NPC1) mRNA levels presented as a fold change. 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.
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