Immunoblot Analysis of Cell Signaling Proteins

Cells were treated with HQGGT, HQ, GG, SM, BS, GC, BI, BE, WO, and WS for 48 h and processed for immunoblot analysis as previously described [12 (link)]. The following primary antibodies (obtained from Cell Signaling Inc. unless noted) were used at the indicated dilutions: anti-TS, 1:2000 (#9045); anti-E2F1, 1:1000 (#3742); anti-GAPDH, 1:10,000 (#5174; #sc-47724, Santa Cruz); anti-p-NDRG1, 1:1,000 (#5482); anti-NDRG1, 1:1,000 (#9408); anti-p-P65, 1:5,000 (#3033); anti-P65, 1:5,000 (#8242); anti-PUMA, 1:1,000 (#4976); anti-p-RB, 1:1,000 (#9307); anti-RB, 1:1,000 (#9313); anti-CHK1, 1:1,000 (#2345); anti-HSP27, 1:1,000 (#2402); anti-CyclinB1, 1:1,000 (#12231); anti-STAT3, 1:5,000 (#9139); anti-MCL1, 1:1,000 (#5453); anti-CDK4, 1:1,000 (#12790); anti-CDK6, 1:1,000 (#3136); anti-CyclinD1, 1:1,000 (#2978); anti-DHFR, 1:1,000 (#610696, BD Biosciences); anti-TK1, 1:1,000 (#40688, Novus Biologicals). Proteins were detected using SuperSignal West Pico substrate (Pierce; Rockford, IL). Quantitation of signal intensities was performed by densitometry on a HP scanner using NIH IMAGEJ software.

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Liu H., Liu H., Zhou Z., Chung J., Zhang G., Chang J., Parise R.A., Chu E, & Schmitz J.C. (2023). Scutellaria baicalensis enhances 5-fluorouracil-based chemotherapy via inhibition of proliferative signaling pathways. Cell Communication and Signaling : CCS, 21, 147.

Publication 2023

anti-TS anti-E2F1 anti-GAPDH anti-NDRG1 anti-P65 anti-p-RB anti-RB anti-CyclinB1 anti-STAT3 anti-MCL1 anti-CDK4 anti-CDK6 anti-CyclinD1 anti-DHFR

Antibodies Biologicals Cdk6 Cells Densitometry Dilutions E2f1 Gapdh Hsp27 Immunoblot analysis Mcl1 Ndrg1 Novus Proteins Puma Stat3

Corresponding Organization :

Other organizations : University of Pittsburgh, UPMC Hillman Cancer Center

Top 5 similar protocols

Variable analysis

independent variables

HQGGT

dependent variables

Levels of TS
Levels of E2F1
Levels of GAPDH
Levels of p-NDRG1
Levels of NDRG1
Levels of p-P65
Levels of P65
Levels of PUMA
Levels of p-RB
Levels of RB
Levels of CHK1
Levels of HSP27
Levels of CyclinB1
Levels of STAT3
Levels of MCL1
Levels of CDK4
Levels of CDK6
Levels of CyclinD1
Levels of DHFR
Levels of TK1

control variables

Incubation time (48 h)

controls

Positive control: None explicitly mentioned
Negative control: None explicitly mentioned

Annotations

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