Lamp2a

Manufactured by Thermo Fisher Scientific

Sourced in United States

LAMP2A is a lysosome-associated membrane protein that functions as a chaperone-mediated autophagy receptor. It facilitates the translocation of cytosolic proteins into the lysosome for degradation.

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4 protocols using lamp2a

Immunofluorescence Analysis of Alpha-synuclein and LAMP2A

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Immunofluorescence analysis was conducted as previously described (Gao et al., 2016). After receiving 24-hour drug treatment, neurons were collected, fixed in 4% paraformaldehyde for 10 minutes, and permeabilized in 0.5% Triton X-100. After washing in PBS and blocking for 10 minutes, cells were incubated overnight at 4°C with primary antibodies: a rabbit polyclonal antibody against lysosomal-associated membrane protein 2A (LAMP2A, 1:200, Cat# 51-2200, RRID: AB_2533900; Thermo Fisher Scientific) and a mouse monoclonal antibody against α-syn (1:200, Cat# MA5-12272, RRID: AB_10978319; Thermo Fisher Scientific). The neurons were rinsed in PBS, cultured with anti-rabbit Alexa Fluor 488–conjugated secondary antibody (green) (1:200, ab150077, RRID: AB_2630356; Abcam) and anti-mouse Alexa Fluor 594–conjugated secondary antibody (red) (1:200, ab150116, RRID: AB_2650601; Abcam) in the dark for 1 hour at 37°C. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA, USA) and observed by fluorescent microscopy (Olympus Corporation). Fluorescence intensity was measured by ImageJ software (NIH) and normalized to fluorescence intensity of the control group.

Gao Q., Chen R., Wu L., Huang Q., Wang X.X., Tian Y.Y, & Zhang Y.D. (2021). Angiotensin-(1–7) reduces α-synuclein aggregation by enhancing autophagic activity in Parkinson's disease. Neural Regeneration Research, 17(5), 1138-1145.

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Antibody Detection in Cellular Processes

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The following antibodies were used in this study: β-actin (Sigma-Aldrich, A5441), ASF1 and NASP (from Genevieve Almouzni), Calreticulin (Cell Signaling Technologies, 2891), FLAG (Sigma, F1804), HIRA (Abcam, ab20655), Histone H2A (Abcam, ab18255), Histone H3 (Abcam, ab1791), Histone H4 (Abcam, ab10158), HSC70 (Abcam, 19136), HSP90 (Abcam, ab13492), Importin 5 (Santa Cruz, sc11369), GAPDH (Santa Cruz Biotech, 365062), LAMP1 (Abcam, ab25630), LAMP2A (ThermoFisher Scientific, 512200), LC3B (Cell Signaling, 2775), TopoI (Santa Cruz, sc32736), VDAC (Abcam, ab15895), Alexa 488 goat anti-mouse IgG (H + L) (ThermoFisher Scientific, A11001), anti-Mouse IgG HRP (Rockland, 610–1302), anti-Rabbit IgG HRP (Rockland, 611–1322), anti-Rat IgG HRP (Rockland, 612-703-002).

Hormazabal J., Saavedra F., Espinoza-Arratia C., Martinez N.W., Cruces T., Alfaro I.E, & Loyola A. (2022). Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control. Nucleic Acids Research, 50(4), 1875-1887.

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Cholesterol Trafficking Quantification

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Cells were seeded on 4-chamber glass slides and grown in 1% FBS DMEM overnight. Treatment with CQ was carried out for 16 hours followed by addition of 1μg/ml CholEsteryl-BODIPY dissolved in DMSO for 4 hours. Cells were fixed using 4% PFA. Immunofluorescent labeling was performed using Lamp2A and Alexa-488 antibodies (Invitrogen, Carlsbad, CA, USA). To quantify CholEsteryl-BODIPY an outline was drawn around each cell (n≥15) and circularity, area and mean fluorescence were measured, along with several adjacent background readings. Total corrected cell fluorescence (TCCF) = integrated density - (area of selected cell × mean fluorescence of background readings) [46 (link)]. The colocalization signal for each condition was measured from four representative fields, n≥15 cells, using ImageJ. The colocalization index represents the Pearson's coefficient (zero is no colocalization and one means perfect colocalization).

Filippakis H., Alesi N., Ogorek B., Nijmeh J., Khabibullin D., Gutierrez C., Valvezan A.J., Cunningham J., Priolo C, & Henske E.P. (2017). Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R. Oncotarget, 8(24), 38099-38112.

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Antibody Sources for Cellular Analysis

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The sources of antibodies used in this study are LAMP-2A (Invitrogen); GAPDH (Abcam); RNase A (Rockland Immunochemicals); HSP90, β-actin, LC3B, and EF-1α (Cell Signaling Technology); hsc70 (Novus Biological); rHN (generated with residues 20–35 of the rHN peptide and no cross-reaction with HN and HNG; GeneTex); and mTOR CST and GFP antibody (Thermo Fisher Scientific). Antibody against HN or HNG was generated by Genscript using residues 7–24 of the HN peptide.

Gong Z., Tasset I., Diaz A., Anguiano J., Tas E., Cui L., Kuliawat R., Liu H., Kühn B., Cuervo A.M, & Muzumdar R. (2018). Humanin is an endogenous activator of chaperone-mediated autophagy. The Journal of Cell Biology, 217(2), 635-647.

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