The following antibodies were purchased: c-Kit (M-14), STAT3 (C-20), STAT5 (C-17), Erk2 (K-23), Jak2 (C-20), cathepsin D (H-75), CD63 (H-93), actin (I-19) and CD28 (H-193) from Santa Cruz Biotechnology; Kit[pTyr719], Akt, Akt[pSer473], STAT5[pTyr694] (D47E7), cleaved caspase-3 and Erk[pThr202/pTyr204] (E10) from Cell Signaling Technology; TGN46, EEA1, Rab11, Tsg101 and TfR from Abcam; calnexin and ubiquitin (FK2) from Enzo; GM130 (35) and AP2α (8) from BD Transduction Laboratories; p85 from Millipore and LAMP1 from Sigma-Aldrich. Anti-phosphotyrosine antibody (4G10) was kindly provided by Dr Toshinori Nakayama (Chiba University). Alexa-fluor 488 anti-Kit (AF488-anti-Kit; 2B8; Biolegend) was used for the experiments in Figs 2b and 5k . The list of antibodies with source and conditions of immunoblotting and immunofluorescence is shown in Supplementary Table 1 . HRP-labelled anti-mouse Ig, anti-rabbit Ig and anti-goat Ig secondary antibodies were purchased from The Jackson Laboratory. AF488-anti-goat IgG, AF568-anti-rabbit IgG, AF647-anti-goat IgG and AF647-anti-mouse secondary antibodies were obtained from Molecular Probes.
Lamp1
Manufactured by Merck Group
Sourced in United States
LAMP1 is a lysosome-associated membrane protein that serves as a marker for lysosomes. It is a type I transmembrane glycoprotein that is primarily localized to the limiting membrane of lysosomes and late endosomes. LAMP1 is involved in the maintenance of lysosomal structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Merck Group (4 mentions)
β actin, by Merck Group (3 mentions)
Cathepsin d, by Santa Cruz Biotechnology (3 mentions)
Anti rabbit ig, by Jackson ImmunoResearch (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Calnexin, by Enzo Life Sciences (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Gapdh, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
P p70s6k, by Cell Signaling Technology (2 mentions)
P ulk1, by Cell Signaling Technology (2 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Tsg101, by Abcam (1 mentions)
Bace 1, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Parp1, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
24 protocols using lamp1
1
Antibodies for Protein Analysis
2
Protein Expression Analysis in Lysed Cells
3
Anticancer Effects of AzalomycinF4a
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The AGS, MKN45, HGC27, SNU1, KATOIII, Hela and HEK293T cells were obtained from the American Type Culture Collection (ATCC). Human GCa cell lines MGC803 were purchased from China Academia Sinica (Shanghai, PR China). The GCa MGC803, AGS, MKN45, HGC27, SNU1, KATOIII cells were cultured in RPMI-1640 medium and Hela cells, HEK293T embryonic kidney cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO2. Antibodies against the following proteins were used with source and dilution ratios indicated: ATG4B (Cell signaling, #13507, 1:1000 and Proteintech, # Cat No. 15131-1-AP, 1:1000); P62 (Sigma, #P0067, 1:1000); LC3 (Sigma, #ABC929, 1:1000; immunofluorescence 1:100); LAMP1(CST, #15665S, 1:100); Snail (CST, #4719, 1:1000); N-cadherin (CST, #4061, 1:1000); C-caspase7 (CST, #12827, 1:1000); PARP-1 (CST, #9542, 1:1000); GAPDH (CST, #2118, 1:1000); Anti-rabbit IgG Fab2 (CST, #4412s, 1:500); Anti-mouse IgG Fab2 (CST, #4409s, 1:500). AzalomycinF4a was isolated from Streptomyces solisilvae HNM30702 and verified by the NMR and HRESIMS data [34 (link)]. Rapamycin (MCE, #HY-10219); BafilomycinA1 (MCE, # HY-100558); Tioconazole (MCE, #HY-1303191 CS-2360); Acridine Orange (Sigma, #MKCD9806); FMK9a (MCE, HY-100522); DAPI (Beyotime, ON.C1005).
4
Antibody Reagents for Cellular Signaling
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The following antibodies were purchased: Kit (M-14), STAT5 (C-17), Erk2 (K-23), Src (Src2) and from Santa Cruz Biotechnology; Kit[pTyr719], Kit[pTyr703], Akt (40D4), Akt[pT308] (C31E5E), STAT5[pTyr694] (D47E7), PDGFRα (D13C6), Erk[pThr202/pTyr204] (E10), golgin97 (D8P2K) and Src[pY416] (D49G4) from Cell Signaling Technology (Danvers, MA, USA); p85, Kit[pTyr568/570], pTyr (4G10) and Src (327) from Millipore; golgin97 (CDF4) and calnexin (AF18) from Thermo Scientific Pierce (Rockford, IL, USA); calnexin from Enzo (Farmingdale, NY, USA); GM130 (35), Yes (1) and Fyn (25) from BD Transduction Laboratories (Franklin Lakes, NJ, USA); LAMP1 from Sigma and Kit (104D2) from Biolegend (San Diego, CA, USA). Horseradish peroxidase-labeled secondary antibodies were purchased from the Jackson Laboratory (Bar Harbor, MA, USA). Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA).
5
Nanoparticle Uptake and Trafficking in Monocyte-Derived Dendritic Cells
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Uptake and intracellular
trafficking of nanoparticles were studied on a monocyte-derived DC
(moDC) culture. Day-3 immature moDCs were collected from the Costar
flasks and counted using Trypan Blue. The cell suspension was diluted
to the desired concentration (0.1 × 106/mL). Then,
0.2 × 105 cells were plated on sterile coverslips
in 24-well plates and were labeled with green NP at a concentration
1 mg of nanoparticles per million cells. The cells were then incubated
for 6 and 24 h at 37 °C. At each time point, the medium was removed
and the coverslips were washed carefully with PBS. The cells were
then fixed by adding 300 μL of 2% paraformaldehyde (PFA) and
permeabilized with PBA + 0.1% Saponin. LAMP1 (Sigma-Aldrich)- or EEA1
(BD Bioscience)-specific primary antibodies (Ab) were incubated with
cells followed by staining with isotype-specific 568AlexaFluor-conjugated
secondary Ab. The cell nuclei were stained with DAPI using 4 μL
droplets of a Mowiol–DAPI mixture. Coverslips were transferred
to the slides, kept overnight in the dark, and then examined with
an Olympus FV1000 confocal laser scanning microscope. An argon laser
(488 nm) was used as the excitation source, and images were collected
with an UPLSAPO 60× objective at a 502–538 nm range. Images
were then processed with ImageJ software.
trafficking of nanoparticles were studied on a monocyte-derived DC
(moDC) culture. Day-3 immature moDCs were collected from the Costar
flasks and counted using Trypan Blue. The cell suspension was diluted
to the desired concentration (0.1 × 106/mL). Then,
0.2 × 105 cells were plated on sterile coverslips
in 24-well plates and were labeled with green NP at a concentration
1 mg of nanoparticles per million cells. The cells were then incubated
for 6 and 24 h at 37 °C. At each time point, the medium was removed
and the coverslips were washed carefully with PBS. The cells were
then fixed by adding 300 μL of 2% paraformaldehyde (PFA) and
permeabilized with PBA + 0.1% Saponin. LAMP1 (Sigma-Aldrich)- or EEA1
(BD Bioscience)-specific primary antibodies (Ab) were incubated with
cells followed by staining with isotype-specific 568AlexaFluor-conjugated
secondary Ab. The cell nuclei were stained with DAPI using 4 μL
droplets of a Mowiol–DAPI mixture. Coverslips were transferred
to the slides, kept overnight in the dark, and then examined with
an Olympus FV1000 confocal laser scanning microscope. An argon laser
(488 nm) was used as the excitation source, and images were collected
with an UPLSAPO 60× objective at a 502–538 nm range. Images
were then processed with ImageJ software.
6
Antibody Validation and Visualization Protocol
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The antibody for Sp56 was kindly gifted from Prof. Fei Gao. Other antibodies were purchased according to the following information: Afaf (Abcam, Cambridge, UK, ab121470); Drp1 (BD bioscience, Franklin Lakes, NJ, USA, 611113); GM130 (Abcam, ab52649); LAMP1 (Sigma-Aldrich, St. Louis, MO, USA, L1418); LC3B (Sigma-Aldrich, L7543); Nrdp1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365622); OPTN (Proteintech, Rosemont, IL, USA, 10837-1-A); Parkin (Sigma-Aldrich, P6248); p62 (Cell Signaling Technology, Danvers, MA, USA, 88588); SIP/CacyBP (Santa Cruz, sc-166455); Sox9 (Abcam, ab185966); Tim23 (BD bioscience; 611222); VAMP8 (Abcam, ab76021); Ubiquitin (Cell Signaling Technology, 3936S); and β-actin (Sigma-Aldrich, A5441). Peroxidase-conjugated anti-mouse IgG (ZSGB-BIO, Beijing, China, ZB-5305, 1:5000), anti-rat IgG (ZSGB-BIO, ZB-2307, 1:4000), or anti-rabbit IgG (ZSGB-BIO, ZB-5301, 1:3000) was used as the secondary antibody. The protein bands were visualized using the ECL detection system (Millipore, Burlington, MA, USA).
7
Quantitative Western Blot Analysis of ALS-related Proteins
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Protein extracts (30μg each) from ALS1 and WT samples were separated by SDS-PAGE and then subjected to Western blotting and immunodetection with the primary antibodies: anti-SOD1, -Cathepsin S, -Cathepsin D, -Cathepsin B (Santa Cruz Biotechnology, CA, USA) [33 (link),41 (link)], -LC3B, (Cell signaling Technology, Danvers, MA, USA), Lamp1 (Sigma Aldrich, St. Louis, MI, USA) and -Actin (Sigma Aldrich, St. Louis, MI, USA), and, with one of the following secondary antibody: Anti-rabbit IgG, HRP-linked Antibody (Cell signaling Technology, Danvers, MA, USA), Anti-mouse IgG, HRP-linked Antibody (Cell signaling Technology, Danvers, MA, USA) Rabbit Anti-Goat IgG Antibody, HRP conjugate (Sigma Aldrich, St. Louis, MI, USA). The ECLTM Detection System (GE Healthcare, Fairfield, CT, USA) was used for the immunostaining procedures. The same blot was re-probed with different antibodies for comparative analyses.
Densitometry analyses were conducted by the FIJI software (FIJI Life-Line version, v.2015, National Institutes of Health, Bethesda, MD, USA). Relative band intensities were normalized to Actin as internal references. Results are expressed as mean ± SD of three independent experiments.
Densitometry analyses were conducted by the FIJI software (FIJI Life-Line version, v.2015, National Institutes of Health, Bethesda, MD, USA). Relative band intensities were normalized to Actin as internal references. Results are expressed as mean ± SD of three independent experiments.
8
Immunofluorescent Localization of ATP13A2 and LAMP-1
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Cells were fixed with 4% paraformaldehyde at 37°C (30 min) and permeabilized with 0.1% Triton X-100 at room temperature (10 min). After blocking in PBS containing 1% BSA and 10% goat serum for 1 h at room temperature, samples were incubated with primary antibodies targeting ATP13A2 or LAMP-1 (Sigma) overnight at 4°C. Thereafter, samples were washed and exposed to Alexa Fluor 488 (green) or Alexa Fluor 647 (red) secondary antibodies. Cells were counterstained with DAPI (1 μg/mL) for 10 min, mounted using Prolong Gold antifade reagent, and cured overnight. Images were acquired with an Olympus IX73 fluorescent microscope using a 63x objective and dimension cellSens software. Scale bars represent 10 μM.
9
Immunofluorescence and Western Blot Antibody Protocol
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The following antibodies were used for immunofluorescence14 and Western blot experiments: LAMP1 (Sigma; #SAB3500285), LC3 (Abcam; #ab62721), β‐Actin (Sigma; #A5441), cathepsin D (SANTA CRUZ; # SC‐377299), Cathepsin L (Abcam; #ab6314), GAPDH (Abcam; #ab9484), TFEB (Sigma; # 3110428), Histone H3 (Cell Signaling; #4499), mTOR1 (Cell Signaling; #2971), p‐mTOR1 (Cell Signaling; #5536), P70S6K (Abcam; #ab32529), p‐P70S6K (Cell Signaling; #9206), AMPK (Cell Signaling; #5831), p‐AMPK (Cell Signaling; #2535), ACC (Cell Signaling; #3676), p‐ACC (Cell Signaling; #11818), ULK1 (Cell Signaling; #8054), p‐ULK1 (Cell Signaling; 5869), DAPI (Sigma; #D9564), Fluoromount™ Aqueous Mounting Medium (Sigma; #SLBN4103V), EX527 (Aladdin Industrial Corporation; #E129892), Resveratrol (Aladdin Industrial Corporation; #R107315).
10
Imaging Monocyte Subcellular Localization
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Fibronectin-coated coverslips were made by incubation of 20 μg/mL Fibronectin (Roche) in PBS for 1 h at 37 °C. Monocytes were adhered on Fibronectin-coated coverslips for 2 h and subsequently fixed with 2 % paraformaldehyde (PFA) and blocked with 3 % bovine serum albumin (BSA), 1 % HS and 10 mM glycine in PBS for 30 min at room temperature (RT). Cells were permeabilized and stained with antibodies against CD53 (mem53, Serotec), CD37 (WR17, home-made), calreticulin (ER marker, Sigma), syntaxin 12/13 [endosome marker, Synaptic Systems (cat. no. 110132)] and Lamp1 (lysosome marker, Sigma-Aldrich) in 0.5 % saponin, 1 % BSA, 10 mM glycine, 1 % HS in PBS, followed by goat-anti-mouse Alexa488 and goat-anti-rabbit Alexa647 (Molecular Probes). Samples were imaged with an Olympus FV1000 confocal laser scanning microscope. Images were analyzed using Fiji software (Schindelin et al. 2012 (link)).
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