Immunoblotting was performed per antibody manufacturer’s specifications. Cells were lysed using Tris Lysis Buffer and Protease inhibitor cocktail (Meso Scale Diagnostics) and immunoprecipitated overnight at 4℃ with Protein A/G Agarose beads (Thermo Fisher) and the following antibodies: anti-MCL-1 (BD Pharmigen); anti-BCL-XL (EMD Millipore); anti-mouse IgG1 isotype control (Cell Signaling). The immunoprecipitate, the supernatant, and a sample of the initial whole cell lysate for each condition were then analyzed by western blot.
Anti mouse igg1 isotype control
Manufactured by Cell Signaling Technology
Anti-mouse IgG1 isotype control is a laboratory reagent used as a control in immunoassays. It is a purified mouse immunoglobulin G1 (IgG1) that does not bind to any known antigenic target, serving as a negative control to establish background signal levels.
Automatically generated - may contain errors
2 protocols using anti mouse igg1 isotype control
1
Immunoprecipitation and Western Blotting
2
ChIP-qPCR of Smad4 in AR-expressing C2C12 cells
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Chromatin immunoprecipitation (ChIP) for Smad4 was conducted on C2C12 cells stably expressing AR24Q or AR100Q. Untransfected MCF7 cells were included as the negative controls. Cells were treated with DHT and BMP7 as previously outlined. Cells were cross-linked with 1% formaldehyde for 10 min, harvested and lysates were sonicated to an average DNA fragment length of 200–400 bp. The experiment was conducted using 300 μg of chromatin and 14 μg of anti-mouse Smad4 (B-8) (Santa Cruz, sc-7966) or anti-mouse IgG1 isotype control (Cell Signaling Technology, 5415 s) antibodies as described previously [75 (link)]. DNA samples were purified and subjected to quantitative real-time PCR with the primers listed in Supplementary Table 2 online resource.
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