A375 human malignant melanoma cells were obtained from the Zhong Qiao Xin Zhou Biotechnology Corporation. The cell line was authenticated by short tandem repeat analysis with GeneMapper software. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37°C in a 5% CO2 air atmosphere. DMEM, FBS, 100× penicillin and streptomycin were acquired from Gibco. Antibodies against the following proteins were used: mGPDH (sc‐390830), NRF2 (sc‐13032, sc‐722), haem oxygenase 1 (HO‐1) (sc‐10789), cyclooxygenase (COX‐2) (sc‐166475) and β‐actin (sc‐47778) (Santa Cruz).
Cyclooxygenase cox 2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Cyclooxygenase (COX-2) is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins, prostacyclin, and thromboxanes. It is involved in the inflammatory response and is implicated in various pathological conditions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Cell lytic m, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Image studio lite 4, by LI COR (1 mentions)
Nitrocellulose sheets, by Cytiva (1 mentions)
β actin, by Merck Group (1 mentions)
β actin, by LI COR (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Survivin, by Santa Cruz Biotechnology (1 mentions)
Musashi 1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
4 protocols using cyclooxygenase cox 2
1
Characterization of A375 Melanoma Cells
2
Western Blot Analysis of Protein Expression
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After treatments, whole cell lysates were prepared using Cell Lytic M (Sigma) and separated using mini-protean TGX precast gels under reducing and denaturing condition (Bio-Rad Laboratories, Milan, Italy). Resolved proteins were transferred onto nitrocellulose sheets (Amersham, Freiburg, Germany), and the resulting membranes were saturated with 5% blocking agent (Amersham, Freiburg, Germany) in Tris-buffered saline (TBS, 20 mmol/L Tris, pH 7.6, 132 mmol/L NaCl)/0.1% Tween 20 for 1 h at room temperature. Blots were then incubated overnight at 4 °C with primary antibodies against endothelial nitric oxide synthase (eNOS) (Merck-Millipore, Darmstadt, Germany), cyclooxygenase (COX)-2 (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (Sigma), followed by a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). An enhanced chemiluminescence (ECL) kit (Amersham, Freiburg, Germany) was used to reveal positive bands, according to manufacturer’s instructions. Bands were analyzed quantitatively using the Image Studio Lite 4.0 software (LI-COR Inc., Lincoln, NE, USA) and normalized to β-actin levels.
3
Western Blot and IHC Antibody Sources
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Primary antibodies for Western blotting and immunohistochemistry were purchased as follows: β-actin, Lamin B, cyclooxygenase (COX-2), nitric oxide synthase (iNOS), p53, cytochrome c, Survivin, epidermal growth factor receptor (EGFR), Ki-67 and cluster of differentiation 31 (CD31) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), phosphorylated signal transducer and activator of transcription 3 (STAT3), γ-H2AX, Bax, B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, cleaved caspase-8, poly (ADP-ribose) polymerase (PARP), lysozyme and Musashi-1 from Cell Signaling Technology, Inc.(Danvers, MA, USA).
4
Quantification of Renal Protein Levels
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The protein level in the renal tubule cells and kidney tissues was determined by Western blotting as previously described15 (link). The sodium dodecyl sulfate (SDS)-PAGE was used to separate equal amount of proteins, and then transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies for GRP78 (#3183), Chop (#2895), Bax (#2772), Bcl-2 (#15071), cleaved caspase-3 (#9664), Sirtuin-1 (Sirt1; #8469), phosphorylated NFκB-p65 (#3033) (Cell Signalling, Danvers, MA, USA), catalase (#ab16731), superoxide dismutase 1 (SOD1; #ab13498), Klotho (#ab203576), CD68 (#ab125212) (abcam, Cambridge, MA, USA), NFκB-p65 (#sc-8008), cyclooxygenase (Cox)-2 (#sc1745), β-actin (#sc-47778) (Santa Cruz, Dallas, TX, USA), inducible nitric oxide synthase (iNOS; #610329) (BD, Franklin Lakes, NJ, USA), and Ly6G (#14-5931-82) (eBioscience, San Diego, CA), and then incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). The signals were detected by using enhanced chemiluminescence substrates (Bio-Rad), and developed with a Fuji Blue X-Ray Film. Protein bands were quantitated by using ImageJ software. The raw data/full blot summary is available in the Supplementary Fig. 5 .
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