LLC-PK1 cells (porcine kidney proximal tubule epithelial cells) purchased from the ATCC were grown in M199 medium (Gibco) supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere containing 5% CO2. ATG5 (-/-) and wild-type MEFs were obtained from the RIKEN BioResource Center (Ibaraki, Japan) and maintained in 10% Dulbecco’s Modified-Eagle Medium (DMEM). Tunicamycin, thapsigargin, 3-methyladenine (3-MA), wortmannin, chloroquine, H202, and TBHP were obtained from Sigma. The caspase 3/7 substrate Asp-Glu-Val-Asp-aminomethyl coumarin (DEVD-AMC) was purchased from Peptide International. ER stress signaling sampler kit (Cat# 9956), mTOR signaling sampler kit (Cat# 9862S), and antibodies to cleaved caspase–3 (Cat # 9661), Atg5 (Cat # 2630), Atg12 (Cat # 4180), and LC3 (Cat # 3868) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to beclin–1 (Cat # 612112) and p62 (Cat # 610832) were from BD-Bioscience (San Diego, CA) and antibodies to β-actin (Cat # sc1616-R) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Antibodies to β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Antibodies to β-actin are a type of laboratory reagent used to detect and quantify the presence of the β-actin protein in biological samples. β-actin is a widely expressed cytoskeletal protein that is often used as a reference or control in various biochemical and molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Atg12, by Cell Signaling Technology (1 mentions)
Llc pk1 cells, by American Type Culture Collection (1 mentions)
Antibodies to cleaved caspase 3, by Cell Signaling Technology (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
Goat anti mouse hrp antibodies, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse hrp conjugate, by Jackson ImmunoResearch (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Caspase glo 3 7 assay system, by Promega (1 mentions)
Lumistar omega microplate luminometer, by BMG Labtech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
9 protocols using antibodies to β actin
1
Autophagy and ER Stress Response in LLC-PK1 Cells
2
Autoantibody Induction by Apoptotic Bodies
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Eight-week-old female NZM and FVB mice were administered three subcutaneous injections of tamoxifen-induced apoptotic bodies or cisplatin-induced apoptotic bodies (20 μg in Incomplete Fruend’s Adjuvant (IFA)), at fortnightly intervals. Control animals received IFA. Sera (at 5 weeks) were assessed for the presence of autoantibodies by ELISA and Western blot. For ELISA, autoantigens (Ro60, RNP A, RNP 68, dsDNA; Arotec Diagnostics Limited) were individually adsorbed onto ELISA plates (1 μg/well) and incubated with pooled sera (diluted 1:1000 in PBS containing 1% BSA and 0.1% Tween-20) for 16 hrs at 4°C. Following further incubation with appropriately diluted goat anti-mouse-HRP antibodies (Jackson ImmunoResearch), enzyme activity was visualized by addition of 3,3′,5,5′-Tetramethylbenzidine (TMB; Invitrogen). For Western blot, nitrocellulose strips (containing moieties in LLC1 lysate, resolved on SDS-PAGE) were incubated with pooled sera, diluted 1:1000 in PBS containing 1% BSA and 0.1% Tween-20. After further incubation with an appropriately-diluted goat anti-mouse HRP conjugate (Jackson ImmunoResearch), enzyme activity was visualized by enhanced chemiluminescence (ECL; Pierce). Antibodies to β-actin (Santa Cruz Biotechnology) were employed to verify equivalence of protein content.
3
Caspase-3/7 Activity Assay and Western Blot
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For the caspase activity assay, cells treated with DZ-CIS for 24 h were measured for caspase 3/7 enzymatic activities by the Caspase-Glo® 3/7 Assay System (Promega, Madison, WI) with the recommended protocol by the manufacturer. Luminescence intensity was acquired using a LUMIstar Omega microplate luminometer (BMG Labtech, BioTek, Winooski, VT). For western blot analysis, our previously reported protocol was used [24 ]. Antibodies to poly ADP-ribose polymerase (PARP), caspase 3, and caspase 9 were from Cell Signaling Technology (Danvers, MA). Antibodies to β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) were used.
4
Protein Isolation and Western Blot
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Proteins from the cells were isolated with RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor. The protein homogenate was centrifuged at 4ºC and 12,000 rpm for 30 minutes. Primary antibodies were incubated on the membranes in Tris-buffered saline with Tween 20 at 4ºC overnight, followed by incubation with secondary antibodies for 2 hours at 37ºC. The florescence was detected with an electrochemiluminescent detection system (UVP Inc., Cambridge, UK) using enhanced chemiluminescence detection reagents. To confirm equal protein loading, the gels were stripped and reprobed with antibodies to β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).
5
Auranofin and Gemcitabine Synergistic Cancer Therapy
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MTT, auranofin, dimethylsulfoxide (DMSO), and Polyethylene glycol (PEG) were obtained from Sigma-Aldrich, St. Louis, MO. Gemcitabine was donated from the pharmacy of The University of Texas, MD Anderson Cancer Center (UTMDACC). Ethyl Alcohol and injectable-H2O were obtained from Pharmaco-AAPER (Brookfield, CT) and Hospira (Lake Forest, IL), respectively. Plasmid pCDH-RFP-Luciferase was obtained from Dr. Mien-Chie Hung, from The Department of Molecular and Cellular Oncology, UTMDACC. Radioimmunoprecipitation protein assay (RIPA), PrestoBlue, and human Vascular Endothelial Growth Factor (VEGF) ELISA kit were purchased from Life Technologies (Grand Island, NY). Bio-Rad protein assay was obtained from Bio-Rad Laboratories Inc. (Philadelphia, PA). Polyvinylidene difluoride (PVDF) membranes and Cremophor EL were bought from Millipore (Billerica, MA). Antibodies to Txnrd1, HIF1α, and PARP were purchased from Abcam (Cambridge, MA). Antibodies to β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Matrigel was obtained from Thermo Scientific, South Logan, UT, and Life Science Technologies, Tewksbury, MA, respectively. Gold analysis standard solutions of Au(III) (1000 mg L-1), hydrochloric acid (HCl) suprapure (36%), and nitric acid suprapure (65%), were purchased from Merck, Germany.
6
Breast Cancer Cell Line Culture Conditions
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Human breast cancer cells MDA-MB-231 and MCF-7 were maintained in DMEM (Hyclone, Cramlington, UK) and T47D in RPMI (Hyclone, Cramlington, UK). All media were complemented with 10% fetal bovine serum (FBS) (Hyclone, Cramlington, UK) and 100 U/ml penicillin/streptomycin (Hyclone, Cramlington, UK). 3-methyadenine and chloroquine (CQ) were purchased from Millipore Analyzer (Millipore, Hayward, CA, USA) and Sigma-Aldrich (Saint-Quentin FAllavier, France), respectively. Antibodies to p62/SQSTMI and cleaved PARP were obtained from Abcam (Abcam, Cambridge, UK). Antibodies to LC3, p21 p27, and pErk1/2(Th202/Th204) and to Phospho-p38 MAPK (Thr180/Tyr182) were obtained from Cell Signaling (USA). Antibodies to γH2AX, p21 (WAFA/Cip1), p27 (Kip1), cyclin D1, PCNA, c-myc, Phospho-Rb (Ser807/Ser811) Beclin-1 and p53 were obtained from Millipore (Millipore, Hayward, CA, USA). Antibodies to p16 were obtained from BD Pharmingen (USA). Antibodies to β-actin were obtained from Santa Cruz Biotechnology, Inc (USA).
7
Investigating Signaling Pathways in Cell Cultures
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4‐phenylpyridine was purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, USA). Dulbecco's Modified Eagle Medium (DMEM), penicillin–streptomycin and fetal bovine serum (FBS) were from Thermo Scientific HyClone (Thermo Scientific HyClone, Logan, USA). Antibodies to β‐actin (1:1000, sc‐517,582) and c‐Src (B‐12) (1:1000, sc‐8056) were purchased from Santa Cruz Biotech (Santa Cruz Biotechnology, CA, USA). Antibodies specific for COX‐2 (1:1000, #12282), c‐Jun (1:1000, #9165), p‐c‐Jun (1:1000, #3270), α/β‐tubulin (1:1000, #2148), p38 MAPK (1:1000, #8690), phospho‐p38 MAPK (Thr180/182) (1:1000, #4511), SAPK/JNK (1:1000, #9252), phospho‐SAPK/JNK (Thr183/Tyr185) (1:1000, #4668), p44/42 MAPK (Erk1/2) (1:1000, #4695), phospho‐p44/42 MAPK (Erk1/2) (Thr202/Thr204) (1:1000, #4370), p‐Akt (S473) (1:1000, #9271), Akt (1:1000, #9272), MKK3/6 (1:1000, #9238), p‐MKK3/6 (1:1000, #9231), MKK4/7 (1:1000, #9152), p‐MKK4/7 (1:1000, #9156), MEK1/2 (1:1000, #9122), p‐MEK1/2 (1:1000, #9121), p‐Src (Y416) (1:1000, #6943), p‐EGFR (Y845) (1:1000, #2231), p‐EGFR (Y1068) (1:1000, #3777), p‐EGFR (Y1045) (1:1000, #2237) and EGFR (1:1000, #4267) were purchased from Cell Signalling Biotechnology (Cell Signalling Biotechnology,). Anti‐lamin B1 (1:10000, ab 16,048) was purchased from Abcam (Abcam).
8
Carbazole Derivatives Modulate AMPK Signaling
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TMZ, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (München, Germany). Antibodies to β-actin (#sc-47778), anti-adenosine monophosphate-activated protein kinase (AMPK; #sc-25792), anti-p-AMPK (#sc-33524), and anti-p-mTOR (#sc-101738) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies to anti-p-Akt (#05-736) and anti-Akt (#05-591) were obtained from Millipore (Bedford, MA, USA). Anti-LC3 antibodies (#NB100-2220) were purchased from Novus Biologicals (Littleton, CO, USA). Primary antibodies were used at a dilution of 1:1,000 in 0.1% Tween-20 and secondary antibodies were used at 1:5,000 dilutions. Acridine orange was obtained from Life Technologies (Rockville, MD, USA). Carbazole derivatives were synthesized in the laboratory of the Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University (Taichung, Taiwan), under the supervision of Dr L.J. Huang. Carbazole derivatives were synthesized and obtained by the laboratory of Dr L.J. Huang.
9
Antibody Reactivity to Apoptotic Bodies
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Reactivity of antibodies in reference reagent sera (Center for Disease Control and Prevention, USA) towards moieties in the six preparations of apoptotic bodies was assessed by Western blot, using standard procedures. Briefly, nitrocellulose strips (containing moieties in apoptotic bodies, resolved on SDS-PAGE) were incubated with individual sera, diluted 1:4000 in PBS containing 1% bovine serum albumin (BSA) and 0.1% Tween-20. After further incubation with an appropriately-diluted goat anti-human IgG + IgM-HRP conjugate (Jackson ImmunoResearch), enzyme activity was visualized by enhanced chemiluminescence (ECL; Pierce). Antibodies to β-actin (Santa Cruz Biotechnology) were employed to verify equivalence of protein content.
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