Mitochondria-enriched and cytosolic fractions were isolated from cortex, hippocampus, cerebellum and olfactory bulb of WT and Wdfy3+/lacZ mice as described before68 (link). Thirty-five µg of proteins were solubilized in SDS sample buffer (Life Technologies, Grand Island, NY) and loaded onto a 4–12% bis-tris gel (Life Technologies) as previously described68 (link). After transferring proteins with an iBlot apparatus (Life Technologies), membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at 20 °C and subsequently probed overnight at 4 °C with the following antibodies: anti-Lamp2 (Abcam, Cambridge, MA; 1:1,000 dilution), anti-LC3 (Novus Biologicals, Littleton, CO; 1:1,000 dilution), anti-Mfn2 (Proteintech, Rosemont, IL; 1:500 dilution), anti-MnSOD (Millipore, Billerica, MA; 1:1,000 dilution), anti Sqstm1 (Cell Signaling Technology, Danvers, MA; 1:500 dilution), anti-Park2 (Abcam; 1:500 dilution), and anti-Pink1 (Novus Biologicals; 1:1,000 dilution). As a loading control, we used anti-β-actin antibody (Sigma, St. Louis, MO; 1:20,000 dilution, 1 h at 20 °C). Secondary antibodies were from LI-COR (Lincoln, NE; 1:10,000 dilution). Membranes were visualized with the use of the Odyssey Infrared Imaging System (LI-COR) and densitometry analysis carried out with ether the Carestream or ImageJ softwares.
Anti lamp2
Manufactured by Abcam
Sourced in United States
Anti-LAMP2 is a primary antibody that recognizes the LAMP2 protein. LAMP2 is a lysosomal membrane protein involved in the transport and fusion of lysosomes. This antibody can be used to detect and study the LAMP2 protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti tom20, by Santa Cruz Biotechnology (3 mentions)
Anti lc3, by Nanotools (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti rab5, by Cell Signaling Technology (2 mentions)
Anti rab7, by Cell Signaling Technology (2 mentions)
Anti gs28, by BD (2 mentions)
Anti gfp, by Santa Cruz Biotechnology (2 mentions)
Anti tsc2, by Cell Signaling Technology (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Blocking buffer, by LI COR (1 mentions)
Sds sample buffer, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Iblot apparatus, by Thermo Fisher Scientific (1 mentions)
Anti lc3, by Novus Biologicals (1 mentions)
Anti mfn2, by Proteintech (1 mentions)
Anti sqstm1, by Cell Signaling Technology (1 mentions)
Anti park2, by Abcam (1 mentions)
24 protocols using anti lamp2
1
Organelle Fractionation and Western Blotting
2
Immunofluorescence Imaging of Prion Protein Localization
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Cells were grown on poly-L-lysine-coated coverslips for 24 hours before
fixation in 4% paraformaldehyde and washed with PBS prior to blocking in 1% FBS,
0.3% Triton X-100. Cells were incubated at 4 °C for
12 hours in blocking buffer with anti-PrP primary antibodies,
i.e. 3F4 and D18 (InPro Biotechnology) monoclonal antibodies. The
following day, cells were incubated for 1 hour with secondary antibodies
conjugated with AlexaFluor. For PrPC cell surface detection,
cells were incubated at 4 °C for 15 min and probed with
3F4 antibody. Cells were permeabilized with 0.2% Triton-X and stained with
AlexaFlour-488 secondary antibody. To detect Thioflavin-S (ThS)-positive
aggregates, transfected and non-transfected cells were fixed with 4% PFA/4%
sucrose/1% Triton X-100 in PBS. For organelle markers, we used anti-Calnexin (ER
marker), anti-EEA1 (early endosome marker), anti-Tfn (recycling endosome
marker), anti-M6PR (late endosomes marker) and anti-LAMP2 (lysosomes marker)
purchased from Abcam. Nuclei were stained with DAPI dye (VECTOR Laboratories).
Images were acquired with a DMIR2 confocal microscope equipped with Leica
Confocal Software (Leica).
fixation in 4% paraformaldehyde and washed with PBS prior to blocking in 1% FBS,
0.3% Triton X-100. Cells were incubated at 4 °C for
12 hours in blocking buffer with anti-PrP primary antibodies,
i.e. 3F4 and D18 (InPro Biotechnology) monoclonal antibodies. The
following day, cells were incubated for 1 hour with secondary antibodies
conjugated with AlexaFluor. For PrPC cell surface detection,
cells were incubated at 4 °C for 15 min and probed with
3F4 antibody. Cells were permeabilized with 0.2% Triton-X and stained with
AlexaFlour-488 secondary antibody. To detect Thioflavin-S (ThS)-positive
aggregates, transfected and non-transfected cells were fixed with 4% PFA/4%
sucrose/1% Triton X-100 in PBS. For organelle markers, we used anti-Calnexin (ER
marker), anti-EEA1 (early endosome marker), anti-Tfn (recycling endosome
marker), anti-M6PR (late endosomes marker) and anti-LAMP2 (lysosomes marker)
purchased from Abcam. Nuclei were stained with DAPI dye (VECTOR Laboratories).
Images were acquired with a DMIR2 confocal microscope equipped with Leica
Confocal Software (Leica).
3
Immunohistochemical Analysis of LAMP-2 and LC3 II
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Immunohistochemical analysis was administered as described previously [37 (link)]. First, 3-mm slices from pretreated myocardium tissue were placed in a bathing solution of 3% H2O2 and 60% methanol PBS for 30 min and then treated with 0.01 mol/L sodium citrate buffer at 95°C in a microwave oven for 13 min. Then, specimens were treated with 5% normal goat serum and 5% bovine serum albumin in PBS. Before each step, sections were rinsed three times in PBS buffer. Incubation with primary Anti-LAMP-2 (Abcam 1:50) and Anti-LC3 II (Santa Cruz,1:100) antibodies was performed in a PBS-based solution of 1% bovine serum albumin for 12 h at 4°C in the recommended dilutions. After rinsing with PBS, sections were incubated with the corresponding secondary antibodies for 30 min at room temperature. A streptavidin/horseradish peroxidase complex was then applied as a detection system (1:100 dilution) for 1 h. Finally, staining was developed with 3, 39-diaminobenzidine tetra-hydrochloride in 0.05 mol/L Tris-HCl buffer. The primary antibody incubation step was omitted in the negative control. All dates in this study were analyzed by software “Image Pro Plus” (Media Cybernetics Corporation, DC).
4
Immunofluorescence Imaging of Organelle Markers
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HeLaClover-PRP4K cells were plated onto glass coverslips in a 6-well plate and treated overnight with 50 μm chloroquine (Sigma) or stimulated with EGF as above. Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min and immunolabeling was carried out as previously described.10 (link) Fluorescent images were captured with a Zeiss Cell Observer spinning-disk microscope (Intelligent Imaging Innovations, 3i, Boulder, CO, USA) using a 1.4 NA 63 × immersion oil objective lens. Images were processed using only linear adjustments in Adobe Photoshop CS5 and Slidebook (3i) software, which was also used for the analysis of mean fluorescence intensity of immunostaining.
Antibodies used for immunofluorescence were used at the manufacturers recommended dilution unless otherwise noted, and include anti-GFP (Abcam, Toronto, ON, Canada, ab13970) (1:2000 dilution), anti-EEA1 (Cell Signaling, #3288) (1:100 dilution), anti-Rab5 (Cell Signaling, #3547) (1:200 dilution), anti-Rab7 (Cell Signaling, #9367) (1:100 dilution), anti-p62 (Cell Signaling, #7695) (1:400 dilution), anti-LAMP2 (Abcam, ab25631) (1:200 dilution), and anti-p-EGFR (Tyr1068) (Cell Signaling, #3777) (1:800 dilution).
Antibodies used for immunofluorescence were used at the manufacturers recommended dilution unless otherwise noted, and include anti-GFP (Abcam, Toronto, ON, Canada, ab13970) (1:2000 dilution), anti-EEA1 (Cell Signaling, #3288) (1:100 dilution), anti-Rab5 (Cell Signaling, #3547) (1:200 dilution), anti-Rab7 (Cell Signaling, #9367) (1:100 dilution), anti-p62 (Cell Signaling, #7695) (1:400 dilution), anti-LAMP2 (Abcam, ab25631) (1:200 dilution), and anti-p-EGFR (Tyr1068) (Cell Signaling, #3777) (1:800 dilution).
5
Antibody Immunodetection Assay Protocol
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The following antibodies were used for the immunoblot and immunofluorescence assays: anti-GFP (Santa Cruz Biotechnology, #sc-9996), anti-Wipi3 (Thermo Fisher Scientific, #PA5-50864), anti-Atg5 (Sigma, #A0731), anti-Lamp2 (Abcam, #ab13524), anti-RFP (MBL, #M204-3), anti-Flag (MBL, #PM020), anti-HA (Santa Cruz Biotechnology, #sc-7392), anti-Lamp1 (abcam, #ab24170), anti-VSVG (KeraFAST, #EB0010), anti-GS28 (BD, #611184), anti-GM130 (BD, #610823), anti-Tom20 (Santa Cruz Biotechnology, #sc-11415), anti-Myc (Santa Cruz Biotechnology, #sc-40), anti-Rab9 (Sigma, #R5404), anti-His (Nacalai Tesque, #04428-26), anti-LC3 (nanoTools, #0231-100), anti-p62 (MBL, #PM045), anti-Wipi2 (abcam, #ab105459), anti-actin (Millipore, #MAB1501), anti-GST (Santa Cruz Biotechnology, #sc-138), anti-Atg7 (CST, #2631), anti-GAPDH (EMD Millipore, #MAB374), anti-calbindin D-28k (Swant, #300), anti-calbindin D-28k (Sigma, #C2724), anti-GFAP (Cell Signaling Technology #12389), anti-Iba1 (Wako, #019-19741), anti-ubiquitin (CST, #3933), anti-ceruloplasmin (BD, #611488), anti-ferritin (abcam, #ab69090), anti-ferroportin (Novus, #NBP1-21502) and anti-DMT1 (Proteintech, #20507-1-AP). Enzymes used for recombinant DNA techniques were purchased from Takara Bio, TOYOBO, and New England BioLabs. All other reagents were obtained from Nacalai Tesque.
6
Quantitative Immunofluorescence Analysis of Lysosomal Proteins
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Protein levels of LAMP2, TFEB, TFE3, MITF, p62, GAPDH, and opsin were assessed using immunofluorescent staining. Seeding of the ARPE-19 cells was similar to that with LysoTracker® Red. Cells were fixed with 4% paraformaldehyde for 15 minutes. Primary antibody was added for 1 hr at room temperature after initial permeabilization and blocking step. Cells were washed twice in dPBS prior to the addition of secondary antibody, Dylight 488 goat anti-rabbit, or Dylight 550 goat anti-mouse and nuclear counterstain, Hoechst dye. Cells were visualized after several washes in PBS. Following antibodies were used: anti-LAMP2 (Abcam, cat. ab25631), -TFEB (Cell Signaling cat. 4240), -TFE3 (Sigma-Aldrich, cat. HPA023881),–MITF (Sigma-Aldrich, cat. HPA003259),-p62 (Cell Signaling cat. 7695) -GAPDH (Cell Signaling cat. 2118), and -opsin (Sigma-Aldrich, cat. O4886). The resultant images (> 6 images or more/well) were automatically captured and quantified using Thermo Fisher Scientific ArrayScan XTI (Thermo Fisher Scientific, Waltham, MA). Quantification of fluorescence intensity was conducted using cell health profiling algorithm, and the result is calculated as fold increase over vehicle control.
7
Western Blot Analysis of ACE2 and Cellular Markers
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Cells were lysed in M2 lysis buffer and sonicated. The protein concentration was determined with a BCA kit (Applygen Technologies Inc., China). Then, the isolated protein was run on an SDS–PAGE gel and transferred to a nitrocellulose membrane. Nitrocellulose membranes were blocked in 5% bovine serum albumin (BSA) and probed with anti-ACE2 (1:1000, Abcam, Cat. ab108252), anti-lamp2 (1:1000, Abcam, Cat. ab25339), anti-GAPDH (1:2000, CST, Cat. 5174), anti-α-Tubulin (1:2000, Sigma–Aldrich, Cat. T6074) and anti-CH25H (1:200, Santa Cruz Biotechnology, Cat. sc-293256) antibodies overnight. Secondary antibodies conjugated to horseradish peroxidase were added, followed by visualization by enhanced chemiluminescence (Thermo Fisher, MA). The results were confirmed by at least three independent experiments.
8
Detecting Ubiquitinated LAMP2 in Cells
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Cells were treated for 4 h with the proteasome inhibitor, MG132 (10 μM), and whole-cell lysates were extracted. The cell lysates were incubated with protein A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h to eliminate non-specific binding, and then with anti-LAMP2 (Abcam, Cambridge, MA, country, ab-125068) overnight at 4 °C. Total lysates were then incubated with protein A for 1 h at 4 °C and centrifuged, and the pelleted proteins were eluted from the beads by being heated in 50 µl of SDS loading buffer for 5 min. Ubiquitinated LAMP2 was detected by Western blot analysis using an anti-ubiquitin antibody (Santa Cruz Biotechnology; sc-8017).
9
PLGA Polymer Materials for Cell Studies
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PLGA‐S‐S‐PLGA (DS‐PLGA) polymer materials (purity, 90%) were purchased from Xi'an Ruixi Biological Technology Co., Ltd. (Xi'an, China). The primary antibodies used in the present study included anti‐BRD4 (1:2000, BETHYL, USA), anti‐c‐Myc (1:1000, CST, USA), anti‐caspase 3 (1:1000, CST, USA), anti‐Cleaved‐caspase 3 (1:1000, CST, USA), anti‐Lamp2 (1:1000, Abcam, USA), and anti‐TTF1 (1:1000, Abcam, USA). DiR (purity > 98%), DiD (purity > 98%), fluorescein isothiocyanate (FITC, purity > 95%), and 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride (DAPI, purity > 98%) were obtained from Thermo Fisher Scientific Inc. (Waltham, USA). The GSH assay kit was provided by Beyotime (Shanghai, China).
10
Multimodal Immunolabelling of Neural Cells
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Cells were fixed with 4% PFA in PBS for 15 min, then blocked and permeabilized with 0.3% Triton-X in bovine serum albumin (BSA, 3%) for 15 min. For LAMP-2 immunolabelling, cells were permeabilized with 0.01% saponin as per manufacturer’s instructions, followed by blocking with 3% BSA for 30 min. Cells were incubated with the following primary antibodies overnight at 4 °C; anti-p75ntr (4 µg/ml; BioLegend); anti-S100β (4 µg/ml; ThermoFisher) and anti-LAMP-2 (4.2 µg/ml; Abcam), anti-MBP (4.3 µg/ml; Gene Tex) The following day, cells were incubated with secondary antibodies donkey anti-rabbit IgG Alexa Fluor 488 (4 µg/ml; Abcam), donkey anti-rat IgG Alexa Fluor 647 (4 µg/ml; Abcam) or donkey anti-rabbit IgG Alexa Fluor 647 (4 µg/ml; Abcam; ThermoFisher). Nuclei were stained using Hoechst 33,342 (1 μg/ml, ThermoFisher). High resolution images were obtained using a confocal microscope (Olympus FV3000).
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