Western Blot Analysis of Protein Signaling

Western blots performed according to published methods^{43 (link),44 (link)}. Polyclonal rabbit antibodies for FLI1, BCL2, SIRT3, SIN3A, cMYC obtained from Abcam, Cambridge, UK; ERK, phospho-ERK, AKT, phospho-AKT, GAPDH, SAP18 antibodies obtained from Cell Signaling Technology (CST), Danvers, MA01923; goat-anti-mouse and goat anti-rabbit HRP-conjugated antibodies obtained from Promega, Madison, Wisconsin, USA. Antibody dilution conducted according to the manufacturer’s instructions. The Oddessy system (Li-Cor Biosciences, Lincoln, USA) used for protein detection. Proteasome (MG132) and transcription (Actinomycin D) and translation (Cycloheximide) inhibitors were obtained from Selleckchem.com and used in some experiments.

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Gajendran B., Varier K.M., Liu W., Wang C., Sample K.M., Zacksenhaus E., Juiwei C., Huang L., Hao X, & Ben-David Y. (2020). A C21-steroidal derivative suppresses T-cell lymphoma in mice by inhibiting SIRT3 via SAP18-SIN3. Communications Biology, 3, 732.

Publication 2020

SIN3A phospho-ERK phospho-AKT GAPDH Oddessy system (Cycloheximide)

Actinomycin d Anti antibodies Antibodies Antibody Bcl2 Cycloheximide Dilution Gapdh Goat Inhibitors Mg132 Mouse Promega Proteasome Protein Rabbit Sirt3 Transcription Western blots

Corresponding Organization :

Other organizations : Guiyang Medical University, University of Madras, Guizhou Provincial People's Hospital, Guizhou University, University of Toronto, First Hospital of Jilin University, Jilin University, Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences

Top 5 similar protocols

Variable analysis

independent variables

Proteasome inhibitor (MG132)
Transcription inhibitor (Actinomycin D)
Translation inhibitor (Cycloheximide)

dependent variables

Protein expression levels of FLI1, BCL2, SIRT3, SIN3A, cMYC, ERK, phospho-ERK, AKT, phospho-AKT, GAPDH, SAP18

control variables

Antibody dilution according to manufacturer's instructions
Oddessy system (Li-Cor Biosciences) for protein detection

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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