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. 2022 Jan 24;12(4):1738–1755. doi: 10.7150/thno.64148

Figure 2.

Figure 2

Active form of RAB7 in autophagosome fractions and MON1A-CCZ1 GEF activity are decreased in AD cell and mice models. (A-B) Bioinformatics analysis of correlation between expression levels of MON1A and RAB7 mRNA and CDR score in parahippocampal gyrus regions of AD patients and controls from the Mount Sinai Brain Bank (MSBB) cohort. (C-D) GTP-RAB7 in autophagosomes isolated from N2a and N2S cells were determined by GST-R7BD affinity-isolation assay. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control. (E-F) GTP-RAB7 in autophagosomes isolated from WT and 3xTg AD mice were determined by GST-R7BD affinity-isolation assay. Data are quantified as mean ± SEM (n =3). *P < 0.05, **P < 0.01, vs. the relative control. (G-H) N2a and N2S cells were transiently transfected with RFP-Lc3 and GFP-CCZ1. Colocalization of RFP-LC3 and GFP-CCZ1 was visualized under confocal microscope. Quantification data are presented as mean ± SEM, n = 20. *P < 0.05, **P < 0.01, vs. the relative control. Scale bar, 7.5 μm. (I-J) N2a and N2S cells were transiently transfected with RFP-Lc3 and GFP-MON1A. The colocalization of RFP-LC3 and GFP-MON1A was visualized under confocal microscope. Quantification data are presented as the mean ± SEM, n = 20. *P < 0.05, **P < 0.01, ***P < 0.001, vs. the relative control. Scale bar, 7.5 μm. (K) CCZ1-MON1A protein was purified by CCZ1 antibody from N2a or N2S cells, and was then subjected to the GEF assay. (L-M) CCZ1-MON1A protein was purified by CCZ1 antibody from hippocampus of 3xTg AD mice or 5xFAD AD mice, and was then subjected to the GEF assay. (N) N2a cells were transiently transfected with Flag/Flag-CCZ1 and GFP-Lc3, followed by immunoprecipitation (IP) with anti-Flag antibody; IP products were resolved by SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies. (O-P) N2a cells and mice brain tissue lysates were subjected to immunoprecipitation using CCZ1 antibody, and the IP products were resolved by SDS-PAGE and analyzed by immunoblotting with the LC3 and VPS34 antibodies (Q) VPS34-associated CCZ1-MON1A protein was purified by VPS34 antibody from WT or 3xTg AD mouse, then was subjected to the GEF assay. (R) CCZ1-binding VPS34 was purified by CCZ1 antibody from WT and 3xTg AD mice brain, and was subjected to lipid kinase activity assay.