Eomesodermin partners with MITF and PU.1 in myeloid precursors to modulate expression of osteoclast lineage determining transcription factors

Published: 29 November 2018| Version 1 | DOI: 10.17632/wdv72kxgjg.1
Contributor:
Sudarshana Sharma

Description

Immunoprecipitation with FLAG-MITF, HA-PU.1, and V5-EOMES in HEK-293 cells.

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Steps to reproduce

For IP experiments, different combinations of FLAG-MITF, HA-PU.1, and V5-EOMES were transfected into HEK-293 cells. Preparation of cell lysates, IP, and Western blot analysis were as previously described (Carey et al., 2016). Briefly, 200 μg of protein was incubated with 1) anti‐FLAG (Sigma-Aldrich Cat #1804); 2) anti-HA (Cell Signaling Cat #3724); or 3) anti-V5 (Sigma-Aldrich Cat #V8012) antibody overnight at 4°C while shaken. Immunoprecipitated proteins were recovered, washed in lysis buffer, and immunocomplexes released with 2X SDS sample buffer.

Institutions

Medical University of South Carolina

Categories

Immunoprecipitation, Western Blot

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