Anti mouse and anti rabbit horseradish peroxidase hrp conjugated secondary antibodies

AZD1480 was from MedChemExpress (Monmouth Junction, NJ, USA) and was dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO, USA). The stock solution (40 mM) was stored at −20 °C. The 35~40% formaldehyde and Triton X-100 were from Sigma. Muse Cell Analyzer was from Merck Millipore (Billerica, MA, USA). FlowJo software to analyze raw data from Muse Cell Analyzer was from FlowJo LCC (version 10.5.3, Ashland, OR, USA). Primary antibodies in this study were as follows: anti-β-actin, anti-GAPDH, anti-Lamin A/C, anti-IL4Rα, and anti-IL13Rα1 were from Santa Cruz Biotechnology, and anti-cleaved PARP-1, anti-cleaved caspase-3, anti-FOXO3, anti-p27, anti-Bax, anti-Bcl-2, anti-JAK2, and anti-pJAK2 were from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies were from Santa Cruz Biotechnology.

Antibodies against human CXCR1 (sc-7303), CXCR2 (sc-7304), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), p65 NFκB (sc-372), IκBα (sc-371), and histone H3 (sc-8654) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against lactate dehydrogenase (LDH; 20-LG22) was from Fitzgerald Industries International (North Acton, MA, USA), and actin antibody was from Sigma (St Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Bortezomib was from ChemieTek (Indianapolis, IN, USA), and carfilzomib was from ApexBio (Houston, TX, USA). Bay-117082 was purchased from Sigma, and SC514 was from Santa Cruz Biotechnology. All other reagents were molecular biology grade and were from Sigma (St Louis, MO).

To analyze protein expression, cells were lysed in radioimmunoprecipitation assay (RPPA) buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland). The same amount of proteins was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using the iBlot gel transfer device (Thermo Fisher Scientific). The membranes were blotted with appropriate primary antibodies and horseradish peroxidase (HRP)–conjugated secondary antibody to detect the proteins of interest. The monoclonal anti-vimentin antibody 84–1 was produced in a mouse. The anti-RPS6-p235p236, anti-RPS6-p240p245, anti-p70S6K-p371, anti-p70S6K-p389, anti-p70S6K, anti-pERK, anti-ERK, anti-p-mTOR, anti-mTOR, and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibodies were from Cell Signaling Technology (Danvers, MA). The anti-mouse and anti-rabbit horseradish peroxidase (HRP)–conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX).