The following antibodies were used in this study: LC3 (1:1000) (PM036, MBL International), actin (1:2500) (A2066, Sigma Aldrich), LAMP1 (1:2000) (H4A3, Developmental studies hybridoma bank, developed by August, J.T./Hildreth, J.E.K. obtained under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA). The signaling pathway antibodies were purchased from Cell Signaling Technology unless otherwise stated: phospho-AKT (1:100) (9271), AKT (1:1000) (9272), phospho-MTOR (1:500) (2974), phospho-RPTOR (1:500) (2083), MTOR (1:1000) (2983), RAB5 (1:1000) (3547S), ERM (1:1000) (3142S), cleaved caspase-3 (1:100) (9664), SQSTM1 (1:500) (MABC32, EMD Millipore), DNM1/dynamin-1(1:1000) (SAB450066, Sigma Aldrich), RAB7 (1:1000) (ab50533, Abcam), RILP (1:500) (ab54559, Abcam), ATP6V0A1 (1:500) (ab105937, Abcam), PLIN2/adipophilin (1:500–1:1000) (GP40, Progen). The antibody to CRYBA1 has been described previously (1:1000).12 (link) FITC-phalloidin was obtained from Molecular Probes-Invitrogen (F432). Rapamycin (R8781) and bafilomycin A1 (B1793) were purchased from Sigma.
Ab50533
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab50533 is a laboratory equipment product offered by Abcam. It serves as a core function, but the detailed description is not available due to the requirement to maintain an unbiased and factual approach without interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Bafilomycin a1, by Merck Group (2 mentions)
Sc 20011, by Santa Cruz Biotechnology (2 mentions)
Ab3612, by Abcam (2 mentions)
A2066, by Merck Group (1 mentions)
Phospho akt, by Merck Group (1 mentions)
Fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Ab105937, by Abcam (1 mentions)
Pm036, by MBL Life Science (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa fluor 568, by Abcam (1 mentions)
Lamp1, by Santa Cruz Biotechnology (1 mentions)
A23187, by Merck Group (1 mentions)
Pp242, by Merck Group (1 mentions)
Torin 1 4247, by Bio-Techne (1 mentions)
Antibody against tfe3 hpa023881, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Bapta am, by Thermo Fisher Scientific (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
13 protocols using ab50533
1
Autophagy Markers and Signaling Pathways
2
Intracellular Trafficking of BACE1 in Neurons
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Cells were fixed using 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 1% FBS for 1 h. Cells were incubated with primary antibodies overnight at 4 °C and labeled with secondary antibodies at 37 °C for 1 h. Images were acquired using confocal fluorescence microscopy (LSM800, ZEISS). For making frozen sections, mice were anesthetized with sodium pentobarbital and perfused with PBS, followed by 4% paraformaldehyde. Following perfusion, the brain was obtained and post-fixed overnight in fixative solution, then cryoprotected overnight in 30% sucrose in PBS. Frozen brain tissues were embedded in TissueTek OCT compound, then cut into 10 μm sections. The sections were observed under confocal fluorescence microscopy (LSM800, ZEISS). BACE1 (1: 200, 3C1C3; 1:200, ab183612) antibody was purchased from Invitrogen or Abcam. EEA1 (1:200, ab2900) antibody and Rab7 (1:200, ab50533) antibody were purchased from Abcam. LAMP1 (1:100, sc-20011) antibody was purchased from Santa Cruz Biotechnology. Alexa Fluor 568 (1:500) and Alexa Fluor 647 (1:500) secondary antibody was purchased from Abcam.
3
Lysosomal and Calcium Signaling in Autophagy
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In this study, we used the following chemicals from Sigma-Aldrich (St Louis, MO): PP242 (P0037), EBSS (Earle’s Balanced Salt Solution; E2888), thapsigargin (T9033), bafilomycin A1 (B1793), A23187 (C7522), ionomycin (I0634), glycyl-L-phenylalanine2-naphthylamide (GPN, G9512), ML-SA1 (SML0627), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP, C2759). LysoTracker (L7528), Fura-2 acetoxymethyl ester (Fura-2 AM; F1221), BAPTA-AM (B1205), and ProLong™ Gold Antifade Mountant with DAPI (P10144) were obtained from Life Technologies. Torin1 (4247) was purchased from Tocris (Bristol, UK). Antibodies against p-RPS6 KB1/S6K (9234), RPS6KB/S6K (9202), p-RPS6/S6 (2211, 2215), p-ULK1 (6888), LAMP1 (9091), CANX (calnexin) (2679), TFEB (4240), PPP3 CA/calcineurin A (2614), SQSTM1/p62 (39749), and histone (9717) were obtained from Cell Signaling Technology. Antibodies against ACTB/actin (sc-47778), GAPDH (sc-47724), TUBA/tubulin (sc-5286), ITPR1 (sc-271197), ITPR2 (sc-398434), and ITPR3 (sc-7277) were obtained from Santa Cruz Biotechnology. Antibodies against MCOLN1/TRPML1 (ab28508) and RAB7A (ab50533) were obtained from Abcam. Antibody against HA (11867423001) was from Roche Diagnostics. Antibody against TFE3 (HPA023881) was purchased from Sigma-Aldrich. ZFYVE27 (LS-C660718) was from LS-Bio. Antibody against LC3B (NB600-1384) was from Novus.
4
Paraffin-Embedded Tissue Immunohistochemistry
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The fresh tissues were fixed in 4% paraformaldehyde at room temperature for 48 h dehydrated in a graded ethanol series and embedded in paraffin. Then, 5 µm thick sections were dewaxed twice in xylene for 10 min and rehydrated in ethanol (75% ethanol 2 h, 95% ethanol 1.5 h, 95% ethanol 1.0 h, absolute ethyl alcohol 1.5 h and absolute ethyl alcohol 1.0 h) at room temperature. The sections were washed with PBS (pH 7.4) 3 times for 3 min. Then, the slides were boiled in 10 mM/l citrate buffer (pH 6.0) in an autoclave for 3 min for antigen retrieval. Following slow cooling, the sections were blocked by immersion in 3% methanolic peroxide for 15 min at room temperature and incubated with the following primary antibodies: Antibodies against Rab7 (ab50533; 1:500; Abcam, Cambridge, UK) overnight at 4°C, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature (31430; 1:5,000; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After washing with water, the slides were counterstained with hematoxylin for 2 min at room temperature, dehydrated and mounted in resin mountant.
5
Intracellular Trafficking of Protein Cargoes
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Cells growing on coverslips in six-well plates were exposed to HPK or HSi at equivalent protein concentrations (20 μg/well) according to our previously established procedures (52 (link)). Briefly, cells were treated on ice for 1 h to promote receptor binding but not internalization, washed to remove unbound protein, and warmed to 37°C to promote synchronized uptake and intracellular trafficking. Cells were fixed at various time points after warming and then processed for immunofluorescence identification of HPK or HSi using an antibody that recognizes the polyhistidine tag (RGS-His antibody; Qiagen). Antibodies for RAB7 and EEA1 were purchased from Abcam (ab50533 and ab206860, respectively). Samples were imaged using a Leica SPE laser scanning confocal microscope. Acquired images were imported to ImageJ and split into individual channels. Individual cells in selected channels were delineated, and integrated densities were measured for each selected area.
6
Visualizing Mycobacterium tuberculosis Localization
7
KCNQ1, KCNE1, and α1A-AR Protein Expression
8
Extracellular Vesicle Protein Extraction
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For protein extraction, EV-pellets (obtained after ultracentrifugation of a pool of four plasma samples, total 6 ml) were resuspended in 100 μl lysis buffer containing 300 mM NaCl, 50 mM Tris pH 7.4, 0.5% NP-40 and anti-proteases cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 15 μg total protein was submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ponceau staining was performed before blocking with 5% milk. Primary commercially available antibodies used were CD63 1:1,000 (CBL553, Millipore, Billerica, MA, United States), RAB27B 1:250 (HPA019849, Sigma-Aldrich), Flotillin 1:500 (ab41927, Abcam, Cambridge, MA, USA), HSP70 1:1,000 (EXOAB-Hsp70A-1, System Biosciences, Mountain View, CA, USA), TSG101 1:500 (ab4A10, Abcam, Cambridge, MA, USA) and RAB7A 1:1,000 (ab50533, Abcam, Cambridge, MA, USA).
9
Investigating Adiponectin Receptor Signaling
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We used the following commercially available antibodies: The anti-AdipoR1 (ab126611), anti-FN (ab2413), anti-Collagen I (ab34710), anti-AMPKα1 + AMPKα2 (ab131512), anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) (ab23875), anti-RAB7(ab50533) and anti-ADRP (ab52356) antibodies were from Abcam; the anti-phospho-AMPKα(Thr172) (2535), anti-mTOR(2972), anti-phospho-mTOR(Ser2448)(5536), anti-ULK1 (8054), anti-phospho-ULK1 (Ser555) (5869), anti-SQSTM1/p62(5114) and anti-LC3B (3868) antibodies were from Cell Signaling Technology; and the anti-SQSTM1/P62 (18420-1-AP), anti-LC3B (18725-1-AP), anti-Beclin1 (11306-1-AP), anti-ADRP (15294-1-AP), anti-ATG5 (10181-2-AP), anti-TFEB (13372-1-AP), anti-β-actin (60008-1-Ig), anti-GAPDH (60004-1-Ig) and anti-Lamin B(12987-1-AP) antibodies were from Proteintech. The anti-SREBP-1c(sc-13551) was from Santa Cruz Biotechnology. AdipoRon and CQ was obtained from Selleckchem. AICAR was provided by Apexbio Technology, SBI-0206965 was purchased from Sigma-Aldrich.
10
Encapsulation and Conjugation of Doxorubicin
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All cells (5 × 103 cells/well) were seeded in 96-well plates and cultured overnight. The media was replaced with drug-supplemented (e.g., encapsulation-DOX, covalent conjugation-DOX and PEG coat-DOX) media at 100 μl/well and incubated for the indicated time. Cells were fixed with 4% paraformaldehyde in medium overnight at 4 °C. Permeabilization was performed in PBS with 0.1% Triton X-100 for 15 min at RT. After a blocking period of 2 hrs with 1% BSA in PBS, cells were incubated with RAB2 (polyclonal, ab131568, Abcam), RAB7 (polyclonal, ab50533, Abcam), RAB9 (polyclonal, ab179815, Abcam) and RAB11 (polyclonal, ab3612, Abcam) antibodies overnight at 4 °C in the absence of light. After washing twice times in PBS, cells were incubated with Alexa Fluor 405 Goat Anti-Rabbit IgG (H + L) and Alexa Fluor 405 Goat Anti-Mouse IgG (H + L) (1:200, A11008, A21057, Molecular Probes) for 2 hrs at RT. Cells were washed twice times in PBS and fluorescence was measured with a microplate reader (VICTOR X3, PerkinElmer, Waltham, MA, USA) at 405 nm (excitation) and 460 nm (emission).
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