![]() 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. Here is a detailed description to quantitate the the bands of western blot bands. (B) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. University of Arkansas for Medical Sciences. 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. This video will help you to quantify your protein bands from western blot. With a simple programing you can set up the macro to be semi or fully automatic. Cholesterol (filipin staining, white) and NPC1 (green). All Answers (3) You can start macro recorder (Plugins>Macros>Record) and record your image manipulations. (B) Western blot of SH-SY5Y control and PADK treated cells using LC3, ABCA1 and NPC1 antibody. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. The Western blot analysis is a powerful tool for protein detection. Student t-test was used for statistical analysis. In standard, qualitative Western blots, the presence or absence of a protein is verified, the molecular weight of the protein is estimated by comparison with known molecular weight markers, and the amount of protein contained in the sample can be estimated by visual 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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