HEK293 was originally purchased from American Type Culture Collection (Manassas, VA, USA). Mouse embryonic fibroblasts (MEFs) were extracted from NIH mouse embryos as report [23 (link)]. Cells were cultured using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), and at 37° C in 5% CO2. Rapamycin (HY-10219) and Stat-3 specific inhibitor (HO-3837, HY-100453) were ordered from MedChemexpress (Monmouth Junction, NJ, USA). Primary antibodies against Runx-2 (sc-390351), OPN (sc-21742), p-Akt1/2/3 (sc-377556), Akt1/2/3 (sc-56878), p-mTOR (sc-517464), and mTOR (sc-293089) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA); primary antibodies against IL-6 (21865-1-AP), Rictor (27248-1-AP), and Raptor (20984-1-AP) were ordered from Proteintech (Wuhan, China); primary antibody against β-actin (AC026) was ordered from ABclonal technology (Wuhan, China).
Rapamycin hy 10219
Manufactured by MedChemExpress
Sourced in United States, China
Rapamycin (HY-10219) is a macrolide compound isolated from the bacterium Streptomyces hygroscopicus. It functions as an inhibitor of the mechanistic target of rapamycin (mTOR) protein, which is a serine/threonine protein kinase that regulates cell growth, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine hy 17589a, by MedChemExpress (4 mentions)
Ab192890, by Abcam (4 mentions)
Cycloheximide s7418, by Selleck Chemicals (3 mentions)
Ab109012, by Abcam (3 mentions)
Deferoxamine mesylate hy b0988, by MedChemExpress (3 mentions)
Rabbit anti β tubulin 2128 antibody, by Cell Signaling Technology (3 mentions)
Rabbit anti tfrc 10084 2 ap, by Proteintech (3 mentions)
Mouse anti flag dykddddk tag, by Abmart (3 mentions)
Lamp1 antibody sc 20011, by Santa Cruz Biotechnology (3 mentions)
Alexa fluor 488 a 21202, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 a 21207 conjugated secondary, by Thermo Fisher Scientific (3 mentions)
Hydrogen peroxide solution 88597, by Merck Group (3 mentions)
Dmso d2650, by Merck Group (3 mentions)
Ab213656, by Abcam (3 mentions)
Ferrostatin 1 hy 100579, by MedChemExpress (3 mentions)
N acetyl l cysteine a9165, by Merck Group (3 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Sc 390351, by Santa Cruz Biotechnology (1 mentions)
Sc 377556, by Santa Cruz Biotechnology (1 mentions)
Sc 56878, by Santa Cruz Biotechnology (1 mentions)
23 protocols using rapamycin hy 10219
1
Osteogenic Differentiation of HEK293 and MEFs
2
Autophagy Regulation Pathway Assay
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The antibodies used in the present study are listed in table S1. Protein A+G agarose beads, LPS, recombinant murine IFN-γ, IL-4, and LysoTracker Red were obtained from Beyotime Biotechnology (Haimen, China), and the Ni2+-NTA agarose was purchased from QIAGEN (Dusseldorf, Germany). CHX was obtained from Calbiochem (508739; Darmstadt, Germany). Dimethyl sulfoxide (DMSO; D2650), MG-132 (C-2211), ammonium chloride (NH4Cl; A9434), 3-MA (M9281), CQ phosphate (PHR1258), and puromycin (P9620) were purchased from Sigma-Aldrich, China. Rapamycin (HY-10219) was obtained from MedChemExpress (Shanghai, China), Bafilomycin A1 (S1413) was purchased from Selleck (Houston, TX, USA), and EBSS was purchased from Gibco (Gaithersburg, MD, USA). A set of small interfering RNA of control (6568) and BECN1 (6246) was purchased from Cell Signaling Technology (Danvers, MA, USA). All other general reagents are commercially available and used as received.
3
Cell Line Maintenance and Authentication
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All cell lines used in this study were obtained from American Type Culture Collection (ATCC) and were authenticated by ATCC. Cells were expanded and preserved in liquid nitrogen upon receipt. Cells for experiments were passaged for fewer than 25-30 times. MDA-MB-361, MDA-MB-231, MDA-MB-157, MDA-MB-435, MDA-MB-468, T47D, SK-BR3, MCF7, BT20, HEK293, 293T and BOSC cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO2. BT474, AU565, ZR75-30 and ZR75-1 were maintained in RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin. MG132 was purchased from Cayman. Chloroquine (CQ) and cycloheximide (CHX) were purchased from Sigma-Aldrich. Rapamycin (HY-10219) was purchased from MedChem Express.
4
Bovine Collagen and mTOR Signaling
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Bovine type II collagen (CII; Chondrex, Inc., Redmond, WA, USA), curcumin (HY-N0005) and rapamycin (HY-10219) (MedChemExpress, Princeton, NJ, USA), were used in this study. Primary antibodies used in this study were all purchased from Cell Signaling Technology (Danvers, MA, USA) as follows: monoclonal anti-4E-BP1 antibody (9644), monoclonal anti-mTOR antibody (2983), monoclonal anti-p70 S6 kinase antibody (2708) and monoclonal anti-Akt1 antibody (75629).
5
Acute Effects of LY294002 and Rapamycin
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LY294002 (HY-10108) or rapamycin (HY-10219) were obtained from MedChem Express (MCE, New Jersey, USA). Male mice at the age of 4–8 weeks were received LY294002 (25 mg/kg body weight)23 (link),24 (link) and rapamycin (10 mg/kg body weight)25 (link) or an equal volume of vehicle by intraperitoneal injection (i.p.) once/day for 2 consecutive days. The behavioral tests or tissue collection were performed at 60 min after the second administration. Each mouse only received injection of one drug (saline, LY294002 or rapamycin).
6
Monoclonal Anti-PEDV N Antibody Protocol
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The monoclonal anti-PEDV N antibody was stocked in our laboratory (48 (link)). Anti-Flag-tag antibody (F1804), chloroquine phosphate (CQ; PHR1258), 3-Methyladenine (3-MA; M9281), and MG132 (M7449) were obtained from Sigma-Aldrich. Antibodies against ACTB/β-actin (66009-1-lg), LC3 (14600-1-AP), NDP52 (12229-1-AP), MARCHF8 (14119-1-AP), FUBP3 (10623-1-AP), GST-tag (10000-0-AP), GAPDH (60004-1-lg), and TRAF3 (66310-1-lg), along with HRP-labeled anti-mouse (SA00001-1) and anti-rabbit (SA00001-2) IgG antibodies could be acquired from Proteintech Group. Bafilomycin A1 (Baf A1; 54645), anti-pIRF3 antibody (4947), anti-pTBK1 antibody (5483), anti-MYC-tag antibody (2278), and anti-HA-tag antibody (3724) were bought from Cell Signaling Technology. Human MARCHF8 siRNA (SC-90432), human NDP52 siRNA (SC-93738) as well as an anti-ubiquitin antibody (SC-8017) were acquired from Santa Cruz Biotechnology. In addition, Rapamycin (HY-10219) was bought from MedChemExpress.
7
Establishing Radioresistant Glioblastoma Cell Lines
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Human glioblastoma cell lines of U251 (TCHu 58) and T98G (CRL 1690) were purchased from Cell Bank of Chinese Academy of Science. The stable radioresistant U251R cell line was developed from its parental cell line U251 by exposing it with 2 Gy X-ray/day for 30 fractions (5 fractions/weekly in general) with a total dose of 60 Gy. Human astrocyte HA1800 cells (C1244) and human microglia HMC3 cells (C1026) were kindly provided by Shanghai WHELAB Bioscience Limited. U251, U251R and HA1800 cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, C11995500BT), while T98G and HMC3 cells were cultured in Minimum Essential medium (Thermo Fisher Scientific, C12571500BT). The above media contained 10% fetal bovine serum, 100 units/mL penicillin and 100 mg/mL streptomycin (Yeasen Biotechnology, 40130ES76). Cells were incubated in a 5% CO2-humidified incubator at 37°C. Cells in log-phase were irradiated with a dose rate of 0.883 Gy/min X-ray by X-Rad320 irradiator (PXI Inc., Santa Rosa, CA, USA). For regulation of autophagic activity, rapamycin (HY-10219), bafilomycin A1 (HY-100558) and chloroquine (HY-17589A) from MedChemExpress were employed.
8
Preparation and Activation of Immune Cells
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LPS (E. coli O55:B5) and β-glucan (CAS: 9041-22-9) were purchased from Sigma (Shanghai, China) and prepared in double distilled water (ddH2O). M-CSF (Novoprotein, Shanghai, China) for BMDM was prepared in ddH2O. Rapamycin (HY-10219), Torkinib (HY-10474) and WAY600 (HY-15272) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA) and prepared in DMSO. The antibodies used for flow cytometry (supplemental Table S1 ) and PCR Assay plates (Wcgene Biotech, Shanghai, China) were all purchased from fcmacs (Nanjing, China). Superparamagnetic iron oxide nanoparticles (SPIO, Ferumoxytol) was kindly provided by professor Ning Gu from Southeast University.
9
Transgenic Mice for Autophagy Analysis
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B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb (RBRC03743) (Hasuwa et al., 2010 (link)), MAP1LC3B-GFP (RBRC00806) (Mizushima et al., 2004 (link)), and Atg3 heterozygous (RBRC02761) (Sou et al., 2008 (link)) mice were bought from Riken BioResource Center. Mito-Tracker Red (40743ES50) was bought from Yeasen. The anti-MAP1LC3B antibody (Medical and Biological Laboratories Co., PM036) was used at 1:200; Alexa Fluor®488 donkey anti-rabbit IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21206) was used at 1:500; anti-Actin (1:5,000) was obtained from Sigma Aldrich (A5441); anti-ATG3 (1:500) was purchased from Cell Signaling Technology (3415S); anti-PINK1 polyclonal antibody (NOVUS, BC100–494) was used at 1:1,000; Anti-OPTN polyclonal antibody (1:1,000) was bought from Proteintech Group (10837-1-AP); FCCP (C2920) and EBSS (E2888) were obtained from Sigma Aldrich. Rapamycin (HY-10219) and Brefeldin A (HY-16592) were bought from Med Chem Express. Mito-Keima vector was shared by Prof. Chen Quan (Wu et al., 2020 (link)). Tim23 antibody (BD Biosciences, 611223) was shared by Prof. Liu Lei.
10
Pancreatic Cancer Cell Line Cultivation and Drug Treatments
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Typical human pancreatic cancer cell lines, PANC-1, MIAPaCa-2, and BXPC-3, were obtained from Procell Life Science and Technology (Wuhan, China) via certified short tandem repeat (STR) profiling. BXPC-3 cells were cultured in RPMI-1640 medium, while PANC-1 and MIAPaCa-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (VivaCell, Shanghai, China) and 1% streptomycin/penicillin (Beyotime, China) at 37 °C and 5% CO2. RSL3 (GC12431) and erastin (GC16630) were obtained from GlpBio (California, USA), while gemcitabine (HY-17026), ferrostatin-1 (HY-100579), torin1 (HY-13003), rapamycin (HY-10219), and AZD8055 (HY-10422) were obtained from MedChemExpress (MCE) (State of New Jersey, USA).
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