Chloroquine (C6628) was obtained from Sigma-Aldrich. Oxaliplatin (OXA, S1224), and quinacrine (S5435) were purchased from Selleckchem. Torin 1 (2273-5) was ordered from BioVision. Anti-ATF4 (CST 11815S), anti-CHOP antibody (2895), anti-eIF2α antibody (9722), anti-p-eIF2α (Ser51) (9721S), anti-Histone H3 (9715S), anti-ATG5 antibody (12994), and anti-PERK (5683) antibodies were obtained from Cell Signaling Technology (CST). An anti-LC3 antibody (NB100-2220) was obtained from Novus Biologicals. An anti-GAPDH antibody (GTX100118) was ordered from GeneTex. Anti-TFE3 antibody (HPA023881), and anti-Flag (F3165) antibody were ordered from Sigma-Aldrich. Anti-TFEB antibody (303-673A) was ordered from Bethyl Laboratories. Anti-HMGB1 (ab18256), and anti-Calreticulin (ab92516) antibodies were ordered from Abcam. Goat anti-rabbit (111-035-003), and goat anti-mouse (115-035-003) secondary antibodies were products of Jackson ImmunoResearch. Alexa Fluor 594 goat anti-rabbit IgG (A-11012), Alexa Fluor 488 goat anti-rabbit IgG (A-11034), and Alexa Fluor 488 (A-11008) goat anti-rabbit secondary antibodies were ordered from Invitrogen.
Anti lc3 antibody nb100 2220
Manufactured by Novus Biologicals
Sourced in United States
The Anti-LC3 antibody (NB100-2220) is a primary antibody that recognizes the LC3 (Microtubule-associated protein 1A/1B-light chain 3) protein. LC3 is a widely used marker for monitoring autophagy, a cellular process for degradation and recycling of damaged or unwanted cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh antibodies ab9132, by Merck Group (2 mentions)
Anti flag antibody f3165, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Gapdh sc 32233, by Santa Cruz Biotechnology (2 mentions)
Chloroquine c6628, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg a11034, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse 115 035 003, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit 111 035 003, by Jackson ImmunoResearch (1 mentions)
Anti perk 5683, by Cell Signaling Technology (1 mentions)
Anti hmgb1 ab18256, by Abcam (1 mentions)
Hrp conjugated anti rabbit antibody w4011, by Promega (1 mentions)
Hrp conjugated anti mouse antibody w4021, by Promega (1 mentions)
Anti fatty acid synthase, by Cell Signaling Technology (1 mentions)
Anti hsl, by Cell Signaling Technology (1 mentions)
Anti p hsl, by Cell Signaling Technology (1 mentions)
Anti beclin 1 nb110 87318, by Novus Biologicals (1 mentions)
Anti atg5 nb110 53818, by Novus Biologicals (1 mentions)
Anti gapdh antibody sc 25778, by Santa Cruz Biotechnology (1 mentions)
Anti acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
Anti adiponectin, by Cell Signaling Technology (1 mentions)
8 protocols using anti lc3 antibody nb100 2220
1
Quantifying Autophagy Pathway Activation
2
Modulation of Autophagy in Cancer Cells
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Ultrapure water (pH 6.7; Milli-Q, Bedford, MA) was used in all situations throughout the experiments. Sodium tetrachloropalladate (II) (Na2PdCl4), decylamine (DA), 3-Methyladenine (M9281), bafilomycin A1 (B1793), Chloroquine (C6628), MitoTEMPO (SML0737) and VAS2870 (SML0273) were purchased from Sigma-Aldrich Co., LLC. (Shanghai, China). Copper (I) chloride (CuCl), poly(vinyl pyrrolidone) (PVP, Mw≈30000), glucose (α or β form), N,N-dimethylformamide (DMF), and ethanol (C2H6O) were purchased from Sinopharm Chemical Reagent Co. Lid. (Shanghai, China). All chemicals were used as received without further purification. Anti-LC3 antibody (NB100–2220, 1:2000 dilution) were purchased from Novus Biologicals. Anti-GAPDH antibodies (AB9132, 1:10,000 dilution) were from Chemicon.,and anti-ATG5 antibody (sc-133158, 1:500 dilution) were from Santa Cruz Biotechnology. HRP-conjugated anti-rabbit antibody (W4011, 1:10,000 dilution) and HRP-conjugated anti-mouse antibody (W4021, 1:10,000 dilution) were purchased from Promega. The HeLa, 4T1, MCF7 cell lines were purchased from ATCC. MCF-7/MDR (kindly provided by Dr Jun Wang at South China University of Technology) were maintained in the DMEM medium containing doxorubicin.
3
Adipocyte Lipolysis and Autophagy Regulation
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Forskolin (F6886), NH4Cl (A9434) and leupeptin (L2884) were purchased from Sigma–Aldrich (St. Louis, MO, USA). H89 (#371962) and KT5823 (#420321) were from Calbiochem (San Diego, CA, USA). Anti-perilipin 1 (#9349), anti-HSL (#4107), anti-pHSL (Ser552)(#4139), anti-pHSL (Ser554)(#4137), anti-pHSL (Ser650)(#4126), anti-acetyl-CoA carboxylase (#3676), anti-adiponectin (#2789), anti-CCAAT/enhancer-binding protein α (C/EBPα) (#8178), anti-fatty acid binding protein 4 (FABP4) (#3544) and anti-fatty acid synthase (FAS) (#3180), anti-p62/SQSTM1 (#5114s) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-beclin-1 (NB110-87318), anti-Atg5 (NB110-53818), and anti-LC3 antibody (NB100-2220) was purchased from Novus Biologicals (Littleton, CO, USA). Anti-GAPDH (SC-25778) antibody was from Santa Cruz Biotechnology (Dallas, TX, USA).
4
Reagents and Antibodies for Cell Culture
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Regents and antibodies. Cycloheximide (5087390001), MG-132 (474791), wortmannin (W3144), 3methyladenine (M9281), brefeldin A (B5936), CDDP (PHR1624) was purchased from Sigma-Aldrich. LysoTracker Red DND-99 (L7528) and Lipofectamine 3000 (L3000015) were from Invitrogen. Anti-p53 (sc-126, Mouse mAb), anti-ATG5 (sc-133158) and anti-SQSTM1 (sc-28359) antibodies were purchased from Santa Cruz Biotechnology. Anti-ubiquitin (58395S) and anti-K63-polyubiquitin (12930S) antibodies were purchased from Cell Signaling Technology. Anti-LC3 antibody (NB100-2220) was purchased from Novus Biologicals. Anti-GAPDH antibodies (AB9132) was purchased from Chemicon. Goat anti-rabbit (Alexa Fluor 488, A-31566) and goat anti-mouse (Alexa Fluor 647, A-21242) secondary antibodies were from Invitrogen.
Cells. Cell culture reagents were purchased from Gibco (Carlsbad, CA). ES-2 and SK-BR-3 were grown in McCoy's 5A Medium. NCI-H1299 and HUVEC were grown in RPMI 1640 medium. MDA-MB-231, MIA PaCa-2 and HEK 293 were grown in DMEM medium. All mediums were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) except for MIA PaCa-2, and MIA PaCa-2 were supplemented with 10% fetal bovine serum (Gibco), 2.5% horse serum and 1% penicillin-streptomycin (Gibco). All cell lines were from ATCC and were cultured at 37°C under 5 % CO 2 .
Cells. Cell culture reagents were purchased from Gibco (Carlsbad, CA). ES-2 and SK-BR-3 were grown in McCoy's 5A Medium. NCI-H1299 and HUVEC were grown in RPMI 1640 medium. MDA-MB-231, MIA PaCa-2 and HEK 293 were grown in DMEM medium. All mediums were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) except for MIA PaCa-2, and MIA PaCa-2 were supplemented with 10% fetal bovine serum (Gibco), 2.5% horse serum and 1% penicillin-streptomycin (Gibco). All cell lines were from ATCC and were cultured at 37°C under 5 % CO 2 .
5
Cellular Signaling Pathway Analysis
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CHX (01810), MG132 (C2211), CQ (C6628) and anti-Flag antibody (F3165) were obtained from Sigma (St. Louis, MO, USA). Anti-E-cadherin antibody (610181) was purchased from BD Biosciences (San Jose, CA, USA). Anti-vimentin (2870), N-cadherin (5741), HA (3724), Beclin 1 (3738), acetylated lysine (9441) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p62/SQSTM1 (sc-28359) and GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-LC3 (NB100–2220) antibody was obtained from NOVUS (Littleton, CO, USA).
6
Comprehensive Stem Cell Characterization
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Anti-cIAP1 (#7065), Anti-cIAP2 (#3130), Anti-survivin (#2808) , Anti-Oct4 (#2750), Anti-klf4 (#12173), Anti-SUMO1 (#4930), Anti- HA-Tag (#3724), Phospho-p44/42 MAPK (Erk1/2) ( #4377), Cleaved-PARP (#5625) antibodies were purchased from Cell Signaling Technology; Anti-Naong ( ab21624), Anti-Sox2 ((ab92494) were obtained from Abcam; Anti-phospho-Sox2(S251) (AP3742a ) antibody was purchased from Abgent; Anti-LC3 (NB100-2220) antibody was obtained from Novus; Anti-Flag (F3165) and Anti-α-Tubulin (T9026) antibodies were obtained from Sigma; Anti-XIAP(sc-55551), Anti- ERK1/2(sc-292838), Anti-p62/SQSTM1 (sc-28359), Anti-caspase-3(sc-56046), goat anti-mouse IgG-HRP (sc-2005), goat anti-rabbit IgG-HRP (sc-2030) antibodies were purchased from Santa Cruz Biotechnology.
Bafilomycin A1, Chloroquine, MG-132, CHX were purchased from Sigma.
Bafilomycin A1, Chloroquine, MG-132, CHX were purchased from Sigma.
7
Western Blot Analysis of Cellular Proteins
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Cell lysates were prepared in 2× Laemmli sample buffer (1610737; Bio-Rad). Total protein quantity was measured using the Bradford dye (5000001; Bio-Rad) in accordance with the manufacturer’s instructions. The samples were then separated by SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane (1620177; Bio-Rad). After blocking with 4% skim milk (BD Bioscience, 90002–594) prepared in TBST [Tris base (T9200; GenDEPOT, Baker, TX, USA), NaCl (G0610; GenDEPOT), and Tween®® 20 (P7949; Sigma-Aldrich)], the membrane was incubated with the indicated primary antibodies. Anti-BRD4 (ab128874), anti-PEX10 (ab196827), and anti-phospho-ATM (ab81292) antibodies were purchased from Abcam; anti-LC3 (NB100-2220) antibody was purchased from NOVUS Biologicals (Centennial, CO, USA); anti-ATM (GTX70103) was purchased from GeneTex (Irvine, CA, USA); and anti-ACTA1 (MAB1501) was purchased from Sigma. For protein detection, membranes were incubated with HRP-conjugated secondary antibodies (7076S and 7074S; Cell Signaling Technology, Danvers, MA, USA).
8
Pancreatic Cancer Cell Line Cultivation
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Pancreatic cancer cell lines (AsPC-1, BxPC-3, PANC-1, CFPAC-1 and SW1990) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Antibodies against YAP (#14074) and ATG5 (#2630) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-active YAP (ab205270) antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies against p62 (sc-28359) and GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-LC3 (NB100-2220) antibody was obtained from Novus Biologicals (Littleton, CO, USA). Anti-Flag antibody (F3165), cycloheximide (CHX, 01810), verteporfin (SML0534) and chloroquine (CQ, C6628) were obtained from Sigma-Aldrich (St.Louis, MO, USA).
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