Whole cell protein extracts from tissue and cell were denatured, separated on SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking in 5% enhanced blocking agent (GE) in Tris-buffered saline–Tween, membranes were probed overnight at 4°C with either, AR (1;1000; cat# 5153, Cell Signaling), SPOP (1:1000; ab168619, Abcam), or phospho-AR (S81) (1:1000; cell signaling, CA USA). After incubation with the appropriate secondary antibody, results were detected using Western Lightning Chemiluminescence Reagent Plus, according tothe manufacturer’s instructions (ThermoFisher Scientific) and captured on film. Quantitative measurements of Western blot analysis were performed using ImageJ and Graph-Pad software (Prism 7).
Western lightning chemiluminescence plus reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Western Lightning Chemiluminescence Plus reagent is a chemiluminescent detection system used for Western blot analysis. It provides a sensitive and reliable method for detecting and quantifying proteins immobilized on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Annexin 5 propidium iodide kit, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiahiazo z y1 3 5 diphenytetrazoliumromide mtt, by Merck Group (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
Annexin 5 pi kit, by BD (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
His tag, by Cell Signaling Technology (1 mentions)
Anti trx, by Abcam (1 mentions)
Anti jab1, by Santa Cruz Biotechnology (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
5 protocols using western lightning chemiluminescence plus reagent
1
Western Blot Analysis of AR and SPOP
2
Western Blot Analysis of EZH2, E-cadherin, and β-Actin
3
Preparation and Use of MLN4924 in Vitro and In Vivo
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MLN4924 was a gift from Fudan University. For in vitro studies, MLN4924 was dissolved in dimethyl sulfoxide (DMSO) and kept in −20 °C. MLN4924 was dissolved in 10% 2-hydroxypropyl- b-cyclodextrin (HPBCD) for the in vivo studies. The MLN4924 solution was freshly made every week and stored in a dark at room temperature before use.
Cell culture reagents and fetal bovine serum (FBS) were obtained from Gibico (Grand Island, NY, USA). The Oligofectamine reagent and Western Lightning Chemiluminescence Plus reagent was from Thermo Scientific Pierce (Waltham, MA, USA). The Annexin-V/propidiumiodide (PI) kit was from BD Biosciences Franklin Lakes, NJ, USA). Human NPC cells used in this study were stored in our lab and were cultured in DMEM medium containing 10% FBS at 37 °C in 5% CO2.
Cell culture reagents and fetal bovine serum (FBS) were obtained from Gibico (Grand Island, NY, USA). The Oligofectamine reagent and Western Lightning Chemiluminescence Plus reagent was from Thermo Scientific Pierce (Waltham, MA, USA). The Annexin-V/propidiumiodide (PI) kit was from BD Biosciences Franklin Lakes, NJ, USA). Human NPC cells used in this study were stored in our lab and were cultured in DMEM medium containing 10% FBS at 37 °C in 5% CO2.
4
Evaluating Apoptotic Signaling Pathways
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Cell culture medium and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, United States). Antibodies to the following proteins were used: PARP (BD Biosciences, San Jose, CA, United States, Cat#556494); Jab1 (Santa Cruz, CA, United States, Cat#sc-13157); p-Akt (p-Thr308) (Cell Signaling, Danvers, MA, United States, Cat#13038), T-Akt (Cell Signaling, Danvers, MA, United States, Cat#2920) and β-actin (Cell Signaling, Danvers, MA, United States, Cat#3700). Western Lightning Chemiluminescence Plus reagent was from Thermo Scientific Pierce (Rockford, IL, United States, Cat# 34087). Annexin V/PI kit was from BD (Cat# 556547). AZA (Cat# 95054), DMSO (Cat# D2650), and 3(4,5)dimethylthiahiazo(zy1)3,5diphenytetrazoliumromide (MTT) (Cat# M2128) were from Sigma.
5
Protein Expression Analysis in Leukemic Cells
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