Whole-cell lysates for Western blots were extracted with Blue Loading Buffer Pack (Cell Signaling Technology, 7722S). Protein lysates were loaded into Novex 16% Tris-Glycine Mini Gels (Thermo Fisher Scientific, XP99165BOX) and separated by gel electrophoresis at 125V for 4 hours. LIN28B and β-actin were detected using an anti-LIN28B antibody (Cell Signaling Technology, #4196) and an anti-ACTB mouse monoclonal antibody (GenScript, A00702), respectively. More details about the antibodies can be found in the Life Sciences Reporting Summary. The Western blot was imaged with Thermo Scientific myECL Imager (Thermo Scientific, 62236).
Blue loading buffer pack
Manufactured by Cell Signaling Technology
Sourced in United States
The Blue Loading Buffer Pack is a ready-to-use solution designed for preparing protein samples for gel electrophoresis. It contains a dye that provides a visual aid for tracking the progress of the electrophoresis run.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads antibody coupling kit, by Thermo Fisher Scientific (3 mentions)
Novex 16 tris glycine mini gels, by Thermo Fisher Scientific (2 mentions)
Anti lin28b antibody, by Cell Signaling Technology (2 mentions)
Anti actb mouse monoclonal antibody, by GenScript (2 mentions)
Thermo scientific myecl imager, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence ecl kit, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Image lab system, by Bio-Rad (2 mentions)
X ray film, by Kodak (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Hybond p, by GE Healthcare (1 mentions)
Ecl prime detection system, by GE Healthcare (1 mentions)
Tanon 5200multi, by Shanghai Tanon Science & Technology (1 mentions)
Ecl reagent, by Vazyme (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Cell Signaling Technology (1 mentions)
12 protocols using blue loading buffer pack
1
LIN28B Western Blot Protocol
2
Whole Cell Lysate Preparation and Protein Interaction Analysis
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Whole cell lysates were prepared in RIPA buffer (1×PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 mM β-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 1× Roche Complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis, IN), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were centrifuged at 13,400 g for 15 min at 4°C. Then, the supernatant fraction was collected. Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo, 1862634). Equivalent aliquots of protein samples (20 to 40 mg) were loaded and electrophoresed on SDS-PAGE gels and then transferred to polyvinylidene fluoride/PVDF membranes. SDS-PAGE, transferring, and immunodetection were performed as previously described (2). To analyze protein interactions, anti-FLAG antibody was incubated with Dynabeads™ Antibody Coupling Kit (Life Technologies, Invitrogen, CA) according to the manufacturer's instructions, and cell lysates were incubated with beads for 3 h at room temperature. Precipitated proteins were washed with PBS-T for three times, eluted by Blue Loading Buffer Pack (Cell signaling technology, USA), loaded on SDS-PAGE, and immunoblotted.
3
Protein Interaction and Modification Analysis
4
Hypoxia-Induced PDK1 Expression Analysis
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After 24 hours culture under normoxia or hypoxia, 1 × 106 cells of NB4 or THP-1 were lysed in 0.1 ml Blue Loading Buffer Pack (Cell Signaling #7722, Danvers, MA) and 0.02 ml of lysed cells were electrophoresed in 7.5% polyacrylamide gel. The gel was electroblotted onto hybond-P (GE Healthcare, UK). The membrane was hybridized with antibody to PDK1 (mouse monoclonal antibody, IgG1: abcam #ab110335, UK) at 1:1000 dilution and reacted with anti-mouse IgG secondary antibody (Cell Signaling #7076, Danvers, MA) at 1:2000 dilution. The membrane was visualized by ECL-prime Detection System (GE Healthcare, UK). β-actin (horseradish peroxidase-conjugated rabbit monoclonal antibody, IgG: Cell Signaling #5125, Danvers, MA) was also tested as control.
5
Western Blot Analysis of Protein Expression
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Target cells were incubated with the indicated factors for the designated times and subjected to Western blot analysis. Whole-cell lysates were extracted using Blue Loading Buffer Pack (Cell Signaling Technology, Danvers, MA, USA, 7722S) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). Fifty micrograms of mouse liver tissue was homogenized and lysed in RIPA buffer supplemented with 1 mmol/L PMSF to extract total protein. Protein concentrations were determined by BCA protein assay kit and all samples were diluted to the same concentration before denaturation. Sixty micrograms of protein was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, transferred onto a nitrocellulose membrane (NC) membrane by wet transfer (200 mA, 90 min), and incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (all primary and secondary antibodies are presented in Supporting Information Table S1 ). The immunoreactive bands were detected by ECL reagent (Vazyme Biotech, Nanjing, China) using a gel imaging system (Tanon 5200 Multi, Tanon Science & Technology, Shanghai, China). The intensity of the bands was analyzed using the Image J software (NIH, USA).
6
LIN28B Western Blot Protocol
7
Western Blot Analysis of Ataxin 1
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Cells were lysed with Blue Loading Buffer Pack (Cell Signaling Technology, Inc.) with a protease inhibitor cocktail and phenylmethanesulfonyl fluoride (Cell Signaling Technology, Inc.). The protein concentrations were quantified using a BCA Protein Assay Kit (Cell Signaling Technology, Inc.). Total cellular proteins (20 μg) were separated via 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). Bovine serum albumin (BSA; 5%) was used as a blocking agent to reduce background and nonspecific binding. After blocking for 1 h, the membranes were incubated overnight at 4°C with rabbit monoclonal anti-human Ataxin 1 (ATXN1) and rabbit monoclonal anti-human GAPDH (both 1 : 1000; both from Cell Signaling Technology, Inc.) antibodies. GAPDH was used as a loading control for normalization. Following intensive washing, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1 : 1000, Cell Signaling Technology, Inc.) for 1 h at room temperature. The protein bands were visualized using SignalFire™ ECL Reagent with the ImageQuant LAS4000 system (Fujifilm, Tokyo, Japan).
8
Epithelial-Mesenchymal Transition Protein Analysis
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Total protein was extracted from cell monolayers using the Blue Loading Buffer Pack (Cell Signaling Technology) according to the manufacturer's protocol. Equal volumes of each extract were loaded onto a Mini-PROTEAN®-TGX ™ 4-20% precast gel (Bio-Rad, Hercules, CA) and transferred to PVDF (Polyvinylidine Diflouride, GE Healthcare Life Sciences, Pittsburgh, PA). EMT marker expression was probed using antibodies from the CST Epithelial-Mesenchymal Transition Antibody Sampler Kit, and protein size was estimated with the Biotinylated Protein Ladder Detection Pack (Cell Signaling Technology) according to the manufacturer's instructions. Chemiluminescence was detected with SuperSignal West Femto substrate (Thermo Scientific, Waltham, MA) and documented with a FluorChem E system (ProteinSimple, San Jose, CA).
9
Protein Expression Analysis of Dental Cells
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Cells were lysed using the Blue Loading Buffer Pack (Cell Signaling, Danvers, MA, United States ), and the proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, United States ). After blocking with 5% non-fat milk for 60 min, the membranes were treated overnight with a primary antibody against Epfn (Atlas Antibodies, Stockholm, Sweden), KRT14 [MA5-11599 (LL002), Thermo Fisher Scientific], AMGN (Abcam, Cambridge, MA, United States ), ameloblastin [AMBN; sc-50534 (M-300), Santa Cruz Biotechnology, Santa Cruz, CA, United States ], kallikrein-related peptidase 4 (KLK4; Bioworld Technology Inc. Louis Park, MN, United States ), Fst [sc-365003 (C-8), Santa Cruz Biotechnology], phosphorylated Smad1/5/8 (p-Smad1/5/8, Cell Signaling), or β-actin (Cell Signaling) at 4°C. Next, the membranes were treated with the corresponding horseradish peroxidase-labeled secondary antibodies [anti-mouse (sc-516102) and anti-rabbit (sc-2357), Santa Cruz Biotechnology] for 60 min at room temperature. The immunoblots were detected using an Immunostar Zeta kit (Wako). The results of Western blotting were quantified using Image J software and normalized to β-actin and the Dox-minus group.
10
Immunoblotting Protein Expression Analysis
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For immunoblotting, protein samples from cells in each group were collected with Blue Loading Buffer Pack (7722 S, Cell Signaling Technology, Danvers, MA, USA). After heating at 100 °C for 8 min, the protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the PVDF membrane (Millipore, Billerica, MA, USA) to be incubated with the following primary antibodies at 4 °C overnight: anti-phosphorylated-Stat3 (Tyr705, 1:2000; 9145S; Cell Signaling Technology) and anti-β-actin antibody (1:1000; 4970L; Cell Signaling Technology). Next, the membranes were incubated with anti-rabbit or anti-mouse immunoglobulin G conjugated with horseradish peroxidase for 1 h at room temperature. Finally, the ECL detection kit (Beyotime Biotechnology, Shanghai, China) was used to detect the protein bands. The bands were photographed using the UVP BioSpectrum AC image 233 system (Upland, CA, USA).
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