Western Blotting Analysis of BAT Lysates

Whole-tissue BAT lysates were prepared as previously described and quantified by Direct Detect (Millipore) (23 (link)). For blotting NCoR1/2, 50 μg protein lysate was loaded per lane, with each lane representing a separate biological replicate. Ladder used was a prestained high-range multicolor protein ladder (26625; Thermo Fisher Scientific). Wet transfer to PVDF was done overnight, and primary antibodies were applied in 5% BSA overnight, all at 4 °C. Primary antibodies used were anti-SMRTe (06-891; Millipore; 1:1,000), anti-NCoR1 (5948S; Cell Signaling Technology; 1:1,000), and anti-Vinculin (V9264; Sigma; 1:5,000), with appropriate HRP-linked secondary antibodies (Cell Signaling Technology; 1:10,000) applied at room temperature for 1 h. Blots were revealed using either film or a Bio-Rad ChemiDoc.

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Richter H.J., Hauck A.K., Batmanov K., Inoue S.I., So B.N., Kim M., Emmett M.J., Cohen R.N, & Lazar M.A. (2022). Balanced control of thermogenesis by nuclear receptor corepressors in brown adipose tissue. Proceedings of the National Academy of Sciences of the United States of America, 119(33), e2205276119.

Publication 2022

Direct Detect anti-Vinculin (V9264; HRP-linked secondary antibodies ChemiDoc

Antibodies Biological Ncor1 Ncor1 2 Protein Pvdf Replicate Tissue Vinculin

Corresponding Organization :

Other organizations : University of Pennsylvania, University of Chicago

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Variable analysis

independent variables

Protein lysates from whole-tissue BAT

dependent variables

Levels of NCoR1/2 proteins

control variables

Protein concentration loaded per lane (50 μg)
Prestained high-range multicolor protein ladder
Wet transfer to PVDF membrane
Primary antibodies (anti-SMRTe, anti-NCoR1, anti-Vinculin) applied in 5% BSA overnight at 4 °C
HRP-linked secondary antibodies applied at room temperature for 1 hour

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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