Ishikawa cells (~ 3.2 × 106) were treated with 10 nM or 100 nM B[a]P-7,8-dione in a time course (5, 10, 30, 60 and 120 min). Cells were washed in ice-cold PBS and whole-cell extracts were prepared in lysis buffer containing Radioimmunoprecipitation assay buffer (RIPA buffer) (Thermofisher, Waltham, MA) and 1 tablet of protease inhibitor cocktail (Roche, IN). Cells were collected, and debris was pelleted by centrifugation at 15,000 × g for 10 min at 4° C. The supernatants were collected and stored at −20 °C. Total protein (40 μg) was loaded onto 10% (v/v) SDS-PAGE gels, separated, and transferred to nitrocellulose. Membranes were incubated with monoclonal mouse phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9106) or polyclonal rabbit p44/42 MAPK (#9102) (Cell Signaling, Technology Danvers, MA). Bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescent immunodetection with an ECL Detection Kit (GE Healthcare, NJ), and were visualized using the Biorad ChemiDoc imaging system (Hercules, CA). In each case three independent experiments were performed followed by densitometric analysis and one-way ANOVA statistical analysis.
Radio immunoprecipitation assay buffer ripa buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
RIPA buffer is a laboratory reagent used to extract and solubilize proteins from cells and tissues. It is a detergent-based buffer designed to disrupt cell membranes and facilitate the isolation of proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin antibiotic, by Thermo Fisher Scientific (2 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Sds page loading buffer, by Biosesang (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ho 1 antibody, by Abcam (1 mentions)
Lps l2630, by Merck Group (1 mentions)
P nf κb, by Santa Cruz Biotechnology (1 mentions)
P stat1, by Santa Cruz Biotechnology (1 mentions)
P iκb, by Santa Cruz Biotechnology (1 mentions)
Bradford s assay reagent, by Bio-Rad (1 mentions)
Diamidino 2 phenylindole dapi, by Cell Signaling Technology (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
Ezview red anti c myc affinity gel, by Merck Group (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
8 protocols using radio immunoprecipitation assay buffer ripa buffer
1
Benzo[a]pyrene-induced MAPK Activation
2
Anti-inflammatory Activity of Plant Extracts
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Chrysanthemum zawadskii, Peppermint (Mentha piperita) and Glycyrrhiza glabra were purchased from Kyeondong market. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin antibiotics were purchased from Gibco; Thermo Fisher Scientific, Inc. Griess reagent and LPS (L2630) were procured Sigma-Aldrich; Merck KGaA. Quanti-Max™ WST-8 Cell Viability Assay Kit has gotten from Biomax. Goat anti-mouse IgG (H+L) Alexa Fluor plus 488 conjugated secondary antibodies were purchased from Invitrogen; Thermo Fisher Scientific, Inc. Enzyme-linked immunosorbent assay (ELISA) kit PGE2, TNF-α, IL-6, and IL-1β were procured from R&D System. Mouse IFN Beta ELISA Kit (TCM, Serum) was supplied by BL Assay Science. HO-1 activity kit was purchased from Cusabio. Radio-immunoprecipitation assay buffer (RIPA buffer) came from Thermo Fisher Scientific, Inc. Bradford's assay reagent was purchased from Bio-Rad Laboratories, Inc. 5X SDS-PAGE loading buffer was purchased from Biosesang. Antibodies against iNOS, COX-2, p-NF-κB, p-IκB, p-Akt, p-STAT1, STAT1, and β-actin were purchased from Santa Cruz Biotechnology, Inc. HO-1 antibody was procured from Abcam. Horseradish peroxidase (HRP)-IgG secondary antibodies and diamidino-2-phenylindole (DAPI) were purchased from Cell Signaling Technology, Inc.
3
Affinity Purification of GST- and c-myc-Tagged Proteins
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The plasmids encoding the N-terminal Glutathione S-transferase (GST) or c-myc tag linked to the proteins being investigated are listed in Supplementary Table S1 . All GST- and c-myc-tagged proteins were expressed in E. coli Rosetta (DE3). Total proteins containing c-myc fusion proteins (EBF1 or EBF2) were extracted from bacteria by sonication in Radioimmunoprecipitation assay buffer (RIPA buffer;Thermo Fisher Scientific) supplemented with Proteinase Inhibitor Cocktail (Sigma-Aldrich) and purified on EZview™ Red Anti-c-myc Affinity Gel (Sigma-Aldrich) according to the manufacturer’s protocol. Next, the crude extracts containing GST fusion proteins [SLIM1_C1 (aa: 287–428), SLIM1_C2 (aa: 418–568) or SLIM1_Nt (aa: 1–286)] were incubated with above-prepared raisins at 4°C overnight with gentle rocking. The raisins were washed six times with RIPA buffer, boiled with 2× SDS–PAGE gel loading buffer, subjected to SDS–PAGE and next immunoblotted using mouse monoclonal anti-GST IgG and anti-c-myc IgG (Sigma-Aldrich, G1160 and Santa Cruz Biotechnology Inc., SC-40) as primary antibody and anti-mouse IgG conjugated to alkaline phosphatase (Sigma-Aldrich, A3562) as a secondary antibody.
4
Oxidative Stress Modulation in Skin Cells
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Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum were purchased from Gibco; Thermo Fisher Scientific, Inc. Penicillin/streptomycin antibiotics came from Invitrogen; Thermo Fisher Scientific, Inc. EZ-Cytox reagent and EZ-western Lumi Pico Alpha were obtained from DoGenBio. Protease inhibitors, tert-butyl hydroperoxide (tBHP), L-ascorbic acid, and o-toluidine blue were purchased from Sigma-Aldrich. Radio-immunoprecipitation assay buffer (RIPA buffer) was purchased from Thermo Fisher Scientific, Inc. ELISA Kit for Collagen Type I was purchased from Cloud-Clone Corp.. Collagen, elastin, MMP-1, MMP-9, JNK, p-JNK, ERK, p-ERK, p38, p-p38, NF-κB, P-NF-κB, and HRP conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) and actin antibody (Biosciences) were also used in this study.
5
Western Blot Protein Analysis Protocol
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Cells harvested for protein extraction were lysed in Radioimmunoprecipitation Assay buffer (RIPA buffer; Thermo Fisher) supplemented with 1X protease inhibitors (Thermo Fisher). Protein concentration was quantified by Bradford assay (Bio-Rad) following the manufacturer’s protocol. Samples for SDS-PAGE were diluted in 5X Laemmli sample buffer (Bio-Rad) containing 10% β-mercaptoethanol and incubated at 95°C for 10 min. Samples for detection of NPC1 were incubated at 70°C for 10 min to prevent aggregation. Equal amounts of protein were electrophoresed in 10% or 4–20% Mini-Protean TGX gels (Bio-Rad). Following electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad) for immunoblotting. nitrocellulose membranes were incubated with 5% nonfat milk in TBS (50 mM Tris-HCl, pH 7.6; 150 mM NaCl) with 0.1% Tween 20 (TBS-T) and sequentially incubated with primary and secondary antibodies diluted in TBS-T at RT for 1 h. Immunoblot images were captured using an Odyssey CLx imaging system (Li-Cor) and protein bands were quantified using the Image Studio Lite software. Protein expression levels were normalized to GAPDH loading controls.
6
Adipogenesis Induction Protocol
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Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and calf serum (CS) were purchased from HyClone (Logan, UT, USA). IBMX (I7018), dexamethasone (D4902), insulin (I0908) and Oil Red O powder (O0625) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Horse serum (HS), penicillin-streptomycin (PS) 100× solution, penicillin/streptomycin/glutamine (PSG) 100× solution, protease and phosphatase inhibitor cocktails, radioimmunoprecipitation assay buffer (RIPA) buffer, and enhanced chemiluminescence (ECL) western substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
7
Protein Quantification and Western Blot Analysis
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Cells treated with sample extracts were lysed using radioimmunoprecipitation assay buffer (RIPA buffer) (Thermo Fisher Scientific, Waltham, MA, USA) with a 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The protein concentrations in each lysate were quantified using the Bradford assay kit (Bio-Rad, Hercules, CA, USA). Proteins were denatured in sodium dodecyl sulfate (SDS) sample buffer separation with SDS polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) which were blocked with 3% bovine serum albumin in phosphate-buffered saline with tween solution. After 1 h, membranes were incubated with specific primary antibodies and horseradish peroxidase (HRP)-conjugated mouse and rabbit secondary antibodies. The antibodies against Nrf2 (Abcam, Cambridge, UK) and antibodies against β-actin and HO-1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used. To visualize protein bands, ECL Western blotting detection kit (Thermo Fisher Scientific, Waltham, USA) and ImageQuant LAS-4000 (Fujifilm, Tokyo, Japan) were used. The quantification of protein bands was determined with ImageJ software (NIH, Bethesda, MD, USA).
8
Immunoblotting Analysis of IDH Mutations
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The tissue specimens or cell pellets were lysed with Radioimmunoprecipitation assay buffer (RIPA buffer, Thermo Fisher) and quantified using the DC Protein Assay (Bio-Rad). Protein samples were resolved on 4-12% Bis-tris gel (Thermo Fisher) and transferred to PVDF membrane (MilliPore). The membranes were labeled with primary antibodies and visualized through chemiluminescence kit (Bio-Rad). The primary antibodies used in this study include anti-IDH1/2 Mutant (R132/R172), clone MsMab-1 (1:2,000, MilliPore); Flag (1:5,000, Origene); and β-actin (1:5,000, Cell Signaling Technology).
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