Immunoblotting of Protein Extracts

Total proteins were extracted using RIPA buffer containing a protease inhibitor cocktail. After centrifuging at 13,000 rpm for 15 minutes at 4 ℃, the soluble cell extracts were used for immunoblots according to a previous protocol (10 (link)). The primary antibodies included the following: anti-EYA3 (Proteintech, Wuhan, Hubei, China; #21196-1-AP), anti-SIX5 (Invitrogen, Shanghai, China; #PA5-75417), anti-p300 (Santa Cruz Biotechnology, Shanghai, China; sc-48343), anti-GAPDH (Santa Cruz Biotechnology; #sc-47724), anti-Flag (Sigma-Aldrich; #SAB4200071), and anti-Myc (Invitrogen; #R95125). The horseradish peroxidase (HRP)-conjugated secondary antibodies included the following: anti-mouse immunoglobulin G (IgG) (Sigma-Aldrich; #GENA931) and HRP-conjugated anti-rabbit IgG (Sigma-Aldrich; #GENA934).

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Yang C, & Liu H. (2022). Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression. Annals of Translational Medicine, 10(13), 752.

Publication 2022

anti-EYA3 anti-p300 anti-GAPDH anti-Flag anti-Myc anti-mouse immunoglobulin G (IgG) HRP-conjugated anti-rabbit IgG

Anti immunoglobulin Antibodies Buffer Cell extracts Gapdh Horseradish peroxidase Immunoblots Immunoglobulin g Mouse P300 Protease inhibitor Proteins Rabbit Ripa

Corresponding Organization :

Other organizations : Sichuan University, West China Hospital of Sichuan University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of EYA3, SIX5, p300, GAPDH, Flag, and Myc

control variables

Protease inhibitor cocktail used during protein extraction
Centrifugation at 13,000 rpm for 15 minutes at 4 °C
Previous protocol used for immunoblotting (10)

controls

Positive control: GAPDH, a housekeeping protein used to normalize protein expression levels
Negative control: Not explicitly mentioned

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