Western Blot and qRT-PCR Analysis of Signaling Pathways

The protein samples from stimulated or transfected cells were extracted by using the Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., USA). The concentrations of protein samples were quantified by using the BCA™ Protein Assay Kit (Beyotime #P0012). Antibodies purchased from Cell Signaling Technology (Beverly, MA) included: phospho-STAT3 (Tyr705) (#9145), phospho-STAT3 (Ser727) (#49081), STAT3 (#4904), phospho-JAK2 (Tyr1007/1008) (#3771), JAK2 (#3230), phospho-JAK1 (Tyr1034/1035) (#74129), JAK1 (#3344), phospho-TYK2 (Tyr1054/1055) (#68790), TYK2 (#14193), phospho-STAT1 (Tyr701) (#7649), STAT1 (#14994), phospho-AKT (Ser473) (#4060), AKT(Pan) (#4821), phospho-NF-κB p65 (Ser536) (#3033), NF-κB p65 (#8242), cyclin D1 (#2978), cyclin A2 (#4656), cyclin B1 (#12231), Caspase-3 (#29629), Bcl-xL (#2762), and Survivin (#2803). PARP antibody and GAPDH antibody were purchased from Beyotime (#AP102) and Genscript (#A00192), respectively. WB analysis was conducted as previously described^{58 (link)}. Three independent experiments were performed using samples collected at different days. qRT-PCR was conducted as described previously^{59 (link)}. All the primers used were listed in Fig. S1b.

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She S., Zhao Y., Kang B., Chen C., Chen X., Zhang X., Chen W., Dan S., Wang H., Wang Y.J, & Zhao J. (2020). Combined inhibition of JAK1/2 and DNMT1 by newly identified small-molecule compounds synergistically suppresses the survival and proliferation of cervical cancer cells. Cell Death & Disease, 11(9), 724.

Publication 2020

Laemmli Sample Buffer BCA™ Protein Assay Kit phospho-STAT3 STAT3 phospho-JAK2 phospho-JAK1 phospho-TYK2 phospho-STAT1 STAT1 phospho-AKT (Ser phospho-NF-κB p65 Caspase-3 Bcl-xL Survivin GAPDH antibody

Antibodies Antibody Ap102 Assay Caspase 3 Cells Cyclin a2 Cyclin b1 Cyclin d1 Gapdh Jak1 Jak2 Laemmli buffer Nf κb p65 Primers Protein Stat1 Stat3 Survivin Tyk2

Corresponding Organization :

Other organizations : Zhejiang University

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Variable analysis

independent variables

Stimulation
Transfection

dependent variables

Protein expression levels of phosphorylated and total forms of STAT3, JAK2, JAK1, TYK2, STAT1, AKT, NF-κB p65, cyclin D1, cyclin A2, cyclin B1, Caspase-3, Bcl-xL, and Survivin

control variables

GAPDH as a loading control

controls

Positive controls: Not specified
Negative controls: Not specified

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