Protein Expression Analysis of Chidamide and Adriamycin Treated Cells

Kasumi-1 and HL-60/ADM cells were treated with chidamide, adriamycin, or their combination, and cells were lysed in RIPA buffer (Sigma-Aldrich, USA). Protein levels were determined by western blotting as previously described [1 (link), 44 (link)]. Briefly, whole-cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were probed with an appropriate primary antibody and then incubated with a secondary antibody. The immunoblots were visualized using chemiluminescence horseradish peroxidase substrate (Millipore, USA) and analyzed with the Odyssey Infrared Imaging System (LI-COR Biosciences, USA). Antibodies against acetyl-histone H3 (#8173), DNMT3A (#3598), H3K27me3 (#9733), EZH2 (#5246), Smo (#4940), Gli-1 (#2643), AKT (#4685), p-AKT (#9614) and GAPDH (#5174) were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH was used as a loading control.

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Jiang X., Jiang L., Cheng J., Chen F., Ni J., Yin C., Wang Q., Wang Z., Fang D., Yi Z., Yu G., Zhong Q., Carter B.Z, & Meng F. (2021). Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia. Journal of Translational Medicine, 19, 117.

Publication 2021

RIPA buffer chemiluminescence horseradish peroxidase substrate Odyssey Infrared Imaging System GAPDH Horseradish peroxidase-conjugated goat anti-mouse IgG goat anti-rabbit IgG

Adriamycin Anti igg Antibodies Antibody Buffer Cell Chemiluminescence Chidamide Ezh2 Gapdh Goat Histone h3 Hl 60 cells Horseradish peroxidase Immunoblots Membranes Mouse Polyvinylidene difluoride Protein Rabbit Ripa Sds page

Corresponding Organization : Nanfang Hospital

Other organizations : Guhua Hospital, The University of Texas MD Anderson Cancer Center

Top 5 similar protocols

Variable analysis

independent variables

Chidamide
Adriamycin
Combination of chidamide and adriamycin

dependent variables

Protein levels of acetyl-histone H3
DNMT3A
H3K27me3
Gli-1
P-AKT

control variables

Whole-cell lysates
SDS-PAGE separation
Transfer to PVDF membranes
Probing with primary antibodies
Incubation with secondary antibodies
Chemiluminescence HRP substrate visualization
Analysis with Odyssey Infrared Imaging System
GAPDH as loading control

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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