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6 protocols using β actin monoclonal antibody

1

Antibody Dilutions for Immunofluorescence and Western Blot

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SARNP polyclonal antibody (Invitrogen, PA5–56586) was used at a 1:250 dilution in immunofluorescence assay (IFA) and 1:1000 dilution for Western blot assay. β-actin monoclonal antibody (Invitrogen AM4302) was used at 1:5000 dilution in Western blot. SC35 monoclonal antibody (Sigma Aldrich SAB4200725) was used at 1:500 dilution for IFA. SON polyclonal antibody (Genetex GTX129778) was used at 1:500 dilution for IFA.
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2

Antibody Dilutions for Immunofluorescence and Western Blot

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SARNP polyclonal antibody (Invitrogen, PA5–56586) was used at a 1:250 dilution in immunofluorescence assay (IFA) and 1:1000 dilution for Western blot assay. β-actin monoclonal antibody (Invitrogen AM4302) was used at 1:5000 dilution in Western blot. SC35 monoclonal antibody (Sigma Aldrich SAB4200725) was used at 1:500 dilution for IFA. SON polyclonal antibody (Genetex GTX129778) was used at 1:500 dilution for IFA.
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3

Liver Protein Extraction and Western Blot

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The liver tissue was homogenized lysis buffer (1% Triton X-100, 20 mM Tris, 40 mM NaF, 0.2% SDS, 0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 100 mM NaCl, pH 7.5). The extract was centrifuged at 15,000× g for 15 min at 4 °C and then stored at −20 °C before use. Target protein in liver tissue sample was applied for Western blot according to the previous studies [40 (link),41 (link)]. The samples were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes. PPAR-α polyclonal antibody (sc-398394, Santa Cruz Biotechnology Inc., Dallas, TX, USA), acetyl-CoA carboxylase (ACC) monoclonal antibody (3676, Cell Signaling Technology Inc., Danvers, MA, USA), β-actin monoclonal antibody (MA5-15739, Thermo Fisher Scientific, Rockford, IL, USA) were used to determine the protein expression of PPAR-α, ACC, and β-actin by using immunoblotting.
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4

UVB-Induced Apoptosis Pathway Analysis

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Photolyase was applied to the cells for 2 h, followed by UVB irradiation for 2 min and 15 s and culturing for another 6 h. Cell lysates were collected using RIPA lysates (Beyotime, Shanghai, China). Primary antibodies were incubated overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. The following antibodies were used: BCL-2 monoclonal antibody (ThermoFisher Scientific, Cambridge, MA, USA); Bax monoclonal antibody (ThermoFisher Scientific; USA33-6400); caspase 3 monoclonal antibody (ThermoFisher Scientific, 700182); and β-Actin monoclonal antibody (ThermoFisher Scientific, MA1-140).
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5

Investigating PI3K/Akt Pathway in Lung Cancer

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Lung cancer cell line NCI-H446 (Kunming Cell Bank, Chinese Academy of Sciences); methyl thiazolyltetrazolium (MTT), dimethylsulfoxide (DMSO), paclitaxel, lobaplatin and LY294002 (Sigma, St. Louis, MO, USA); RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA); rabbit anti-human PI3K, p-Akt, AKT, phosphorylated-glycogen synthase kinase 3β (p-GSK3β), GSK3β, β-actin monoclonal antibody and goat anti-rabbit secondary polyclonal antibody (cat. nos. 4249, 4060, 4685, 9323, 9315, 4970 and 14708) (Cell Signaling Technology, Danvers, MA, USA); Annexin V-FITC apoptosis assay kit (BD Biosciences, Heidelberg, Germany); inverted fluorescence microscope (Thermo Fisher Scientific, Inc., Waltham, MA, USA); cell culture bottle (Corning Inc., Corning, NY, USA); Transwell chamber (EMD Millipore, Billerica, MA, USA) and pipettor (Eppendorf AG, Hamburg, Germany).
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6

Apoptosis-related Protein Expression

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RPMI1640, DMEM (Dulbecco's Modified Eagle's Medium) culture media and fetal bovine serum were purchased from GIBCO; Parenzyme, SDS, TEMED, β-actin monoclonal antibody, HA monoclonal antibody (Cat. # TA-04), Bcl-2 monoclonal antibody (sc-783) and Bax monoclonal antibody (sc-526) were purchased from Santa Cruz Biotechnology; PUMA polyclonal antibody (Cat. #55120-1-AP) was purchased from USA Proteintech; IgG (ZDR-5307) and IgG (ZDR-5306) were purchased from Beijing ZSGB-Bio Company; DDP cisplatin was purchased from Shandong Qilu Pharmaceutical Co., Ltd.
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