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9 protocols using n ethylmaleimide e3876

1

Cellular Signaling Pathway Investigation

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Nocodazole (M1404), PKH67 (green) (PKH67GL-1KT), PKH26 (red) (PKH26GL-1KT), brefeldin-A (B7651, used at 5 μg/ml), N-ethylmaleimide (E3876, used at 50 μM), LiCl2 (L-4408), PMA (used at 100 nM; P8139), and dynasore (D7693, used at 100 μM) were purchased from Sigma (St. Louis, MO, USA). TNF-α (210-TA) (used at 17 ng/ml), VEGF (used at 19 ng/ml; 293-VE), and neutralizing antibodies to VEGF (used at 1 μg/ml; MAB293) and VEGFR2 (used at 1 μg/ml; MAB3572) were purchased from R&D Systems (Minneapolis, MN, USA). Smac mimetic TL-32711 (A-1901) (used at 100 nM) was purchased from Active Biochem (Maplewood, NJ, USA). Cisplatin (NDC 0015-3220-22) was purchased from Bristol Laboratories (Princeton, NJ, USA). Antibodies to VEGF (for WB: Sc-7269), PKC-ζ (Sc-17781), K-Ras (Sc-30), and VEGFR2 (for WB: Sc-504) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to E-cadherin (3195), p70S6K (2708), phospho-S6 ribosomal protein (Ser-235/236) (2211), S6 ribosomal protein (2317), syntaxin-6 (2869), and cdc42 (2466) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to β-catenin (610153) was purchased from BD Transduction Laboratories (Lexington, KY, USA). BI-D1870 (used at 5 μM; S2843) was purchased from Selleckchem (Houston, TX, USA).
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2

Cellular Pathways Investigated Using Inhibitors

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SD70 (M60194-2s) was purchased from Xcessbio Biosciences Inc; Cycloheximide (CHX; N11534), Calpeptin (C8999), chloroquine (C6628), iodoacetamide (I1149), and N-ethylmaleimide (E-3876) were purchased from Sigma-Aldrich; Sorafenib (8705) was purchased from Cell Signaling Technology; MG-132 (S2619) was purchased from Selleck; Z-VAD-FMK (2163) was purchased from Tocris. Treatment conditions were described in detail in the text.
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3

Comprehensive ZIKV Protein Detection Assay

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Antibodies against Flag (F3165, 1:2000) and HA (H6908, 1:5000) were purchased from Sigma (Burlington, MA, USA). Antibodies against the envelope protein of ZIKV (GTX133314, 1:1000), NS1 protein of ZIKV (GTX133307, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GTX100118, 1:5000), and ZDHHC11 (GTX106800, 1:1000) were purchased from GeneTex (Hsinchu City, Taiwan, P.R.C). For the immunofluorescence studies, antibodies against the envelope protein of flaviviruses (ab214333, 1:100) were purchased from Abcam (Shanghai, China). N-ethylmaleimide (E3876), hydroxylamine hydrochloride (255580), and 2-bromopalmitate (2-BP; 21604) were obtained from Sigma (Burlington, MA, USA). BMCC-biotin (21900) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Streptavidin-horseradish peroxidase (M00091) was purchased from Genscript (Piscataway, NJ, USA). Fluorescein (FITC)-conjugated AffiniPure goat anti-mouse immunoglobulin (Ig)G (H+L) (SA00003-1) and Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) (SA00009-2) were purchased from ProteinTech (Rosemont, IL, USA).
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4

Antibodies for Endoplasmic Reticulum Stress

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Primary antibodies targeting the following proteins were used in this study: inositol‐requiring transmembrane kinase/endoribonuclease (IRE)‐1α (#3294, RRID:AB_823545) and CCAAT‐enhancer‐binding protein homologous protein (CHOP; #2895, RRID:AB_2089254; Cell Signaling Technology, Danvers, MA, USA); 78‐kDa glucose‐regulated protein (GRP78; https://www.scbt.com/scbt/ko/product/grp-78-antibody-76-e6?requestFrom=search, RRID:AB_627698) and β‐actin (https://www.scbt.com/scbt/ko/product/beta-actin-antibody-1?requestFrom=search, RRID:AB_630836; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and phosphorylated p‐IRE‐1α (ab48287, RRID:AB_873899), 4‐hydroxynonenal (4‐HNE; ab48506, RRID:AB_722490), mucin 5AC (ab82423, RRID:AB_1861425), and monoclonal protein disulfide isomerase family A member (PDIA)6 (ab11432, RRID:AB_298038; Abcam, Cambridge, MA, USA). HRP‐conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. IC87114 was from Yuhan Corporation (Seoul, Korea). N‐ethylmaleimide (E‐3876) and dihydroethidium (D7008) were from Sigma‐Aldrich (St. Louis, MO, USA).
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5

Comprehensive Cell Signaling Assay Protocol

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Nocodazole (M1404), PKH67 (green) (PKH67GL-1KT), PKH26 (red) (PKH26GL-1KT), brefeldin-A (B7651, used at 5 μg/ml), N-ethylmaleimide (E3876, used at 50 μM), LiCl2 (L-4408), PMA (used at 100 nM; P8139), and dynasore (D7693, used at 100 μM) were purchased from Sigma. TNF-α (210-TA) (used at 17 ng/ml), VEGF (used at 19 ng/ml; 293-VE), and neutralizing antibodies to VEGF (used at 1 μg/ml; MAB293) and VEGFR2 (used at 1 μg/ml; MAB3572) were purchased from R&D Systems. Smac mimetic TL-32711 (A-1901) (used at 100 nM) was purchased from Active Biochem (Maplewood, NJ, USA). Cisplatin (NDC 0015-3220-22) was purchased from Bristol Laboratories (Princeton, NJ, USA). Antibodies to VEGF (for WB: Sc-7269), PKC-ζ (Sc-17781), K-Ras (Sc-30), and VEGFR2 (for WB: Sc-504) were purchased from Santa Cruz Biotechnology. Antibodies to E-cadherin (3195), p70S6K (2708), phospho-S6 ribosomal protein (Ser-235/236) (2211), S6 ribosomal protein (2317), syntaxin-6 (2869), and cdc42 (2466) were purchased from Cell Signaling Technology. Antibody to β-catenin (610153) was purchased from BD Transduction Laboratories. BI-D1870 (used at 5 μM; S2843) was purchased from Selleckchem (Houston, TX, USA).
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6

Cell Lysis and Protein Extraction

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Cells were washed twice in ice-cold PBS and lysed on ice for 15 min in RIPA buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% Sodium Deoxycholate, 5 mM EDTA, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)). Soluble extracts were obtained by centrifugation at 14,000 × g for 30 min at 4 °C and equal protein amounts were diluted in 2× sample buffer for immunoblot analysis. 20 mM of N-ethylmaleimide (E3876, Sigma) (DUB inhibitor) was added to the RIPA buffer to improve visualization of XPC-ubiquitination bands (after UV)72 (link).
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7

Comprehensive Antibody Library for Stem Cell Research

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Rabbit antibodies against TRIM28 (4124S, 4123S), CTCF (3418S), OCT4A (2890S), SOX2 (23064S), NANOG (3580S), KLF4 (12173S), SUMO2 (4971S), and mouse antibodies against TRA-1-60 (4746T), TRA-1-81(4745T), SSEA-4 (4755T), and SMA (D4K9N) were from Cell Signalling Technology. Anti-SALL4 (ab29112), anti-TRIM24 (ab70560), anti-NOP58 (ab155556), anti-NESTIN (ab196908) were from Abcam. Mouse antibody against α-Tubulin was from Bethyl Laboratories, mouse anti-LaminA/C antibody was from Sigma Aldrich (SAB4200236), rabbit anti-mCherry (PA5-34974), rabbit anti-TRIM33 (PA5-82152) were from Invitrogen, and mouse anti-HIS (34650) was from Qiagen. Rabbit anti-CYTOKERATIN17 was a gift from R. Hickerson (University of Dundee). Sheep antibodies against SUMO1, SUMO2 (27 (link)) were generated in-house. Secondary antibodies conjugated with HRP and Alexa fluorophores, Cy3 Cellmask (C10045), Rhodamine Phalloidin (R415) were from Sigma and Invitrogen, respectively. ML792 (1644342-14-2), MG132 (474787), and N-ethylmaleimide (E3876) were from Sigma Aldrich. Protease Inhibitor cocktail (11836170001) was from Roche. Propidium iodide (P3566) and DAPI (D1306) were from Life Technologies.
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8

Breast Cancer Cell Line Characterization

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The human breast cancer cell lines MCF-7, T47D, Hs578T, and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC); ZR-75B, cloned from the ZR-75-1 cell lines26 (link), was obtained from the National Cancer Institute (NCI)/NIH. The breast cancer cell lines were cultured at 37 °C in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Medium and reagents for cell culture were purchased from WELGENE, Inc., Republic of Korea. In particular, MCF-7 and T47D cells were cultured in DMEM medium without phenol red with 10% dextran-coated charcoal (Sigma)-stripped FBS and 1% penicillin/streptomycin for 24 h prior to 17β-estradiol treatment. The cell lines in this study were not found in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample and were routinely tested for mycoplasma contamination by PCR. Transfections were carried out using Fugene® HD (Promega) or Lipofectamine® RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions. Cycloheximide (01810), 17β-estradiol (E2758), 5-aza-2′-deoxycytidine (A3656), MG132 (C2211), Bafilomycin A1 (B1793), Chloroquine (C6628), 3-methyladenine (M9281) and N-ethylmaleimide (E3876) were purchased from Sigma-Aldrich.
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9

Cytometric Bead Array Immunoassay Protocol

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The cytometric bead array (CBA) polystyrene beads (cat n° 560037), coupling buffer BD TM (cat n° 51-9004756), storage buffer BD TM (cat n° 51-9004758) and wash buffer BD TM (cat n° 51-9003798) were purchased from BD Biosciences. Bovine serum albumin (BSA, A8022), 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-Nhydroxysuccinimide ester sodium salt -sulfo-SMCC (M6035), N-ethylmaleimide (E3876) and dithiothreitol (DTT, 10197777001) were purchased from Sigma-Aldrich. Phosphate buffer saline (PBS) 10x pH 7.2 (70013-032) was purchased from Gibco. Antibodies brilliant ultraviolet (BUV395) mouse anti-human IgG (cat n° 564229), brilliant blue (BB515) mouse anti-human IgM (cat n° 564622), biotin mouse anti-human IgA1/IgA2 (cat n° 555884) and brilliant violet (BV421) streptavidin (cat n° 563259) were purchased from BD Biosciences.
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