Immunoblotting Analysis of Apoptotic Markers

Protein was analyzed by SDS-PAGE as previously described (16 (link)). The following primary antibodies from Cell Signaling Technology were used at manufacturer-recommended dilutions for immunoblotting: cleaved caspase-3 (#9661), total caspase 3 (#9668), cleaved caspase-9 (#9501), total caspase-9 (#9502), phospho-Chk1 (Ser296; #2349), total Chk1 (#2360), phos-pho-Chk2 (Thr68; #2661), total Chk2 (2662), and γH2AX (#9718). β-Actin (Santa Cruz Biotechnology, catalog #sc-47778) was included as a loading control. Species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used at 1:20,000 dilution.

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Zeng L., Beggs R.R., Cooper T.S., N.Weaver A, & S.Yang E. (2017). Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma. Molecular cancer therapeutics, 16(4), 591-600.

Publication 2017

cleaved caspase-3 total caspase 3 cleaved caspase-9 total caspase-9 phospho-Chk1 (Ser296 total Chk1 phos-pho-Chk2 total Chk2 γH2AX β-Actin Species-specific horseradish peroxidase-conjugated secondary antibodies

Actin Antibodies Caspase 3 Caspase 9 Dilution Horseradish peroxidase Protein Sds page

Corresponding Organization : University of Alabama at Birmingham

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Cleaved caspase-3
Total caspase-3
Cleaved caspase-9
Total caspase-9
Phospho-Chk1 (Ser296)
Total Chk1
Phospho-Chk2 (Thr68)
Total Chk2
γH2AX

control variables

β-Actin (loading control)

controls

Positive control: None mentioned
Negative control: None mentioned

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