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FIGURE 17.2
Comparison of standard and membrane co-IP. (A) Schematic summary of standard and
membrane co-IP performed in total cell lysate or on living cells, respectively. (B) Standard
co-IP performed in HEK293T cells transiently transfected with pHA-D 4.2 R and/or pFLAG-
D 4.4 R. Immunoprecipitation (IP) was performed with mouse anti-HA (16B12). After, Western
blot analysis, proteins were visualized with HRP-coupled anti-FLAG M2 or mouse anti-HA
(16B12) and HRP-coupled antimouse. PM-mature, fully glycosylated plasma membrane
receptor; ER-ER-retained receptor.*Signal denoting association of two heavy chains (2
50 kDa) of anti-HA sera. (C) Membrane co-IP was performed in HEK293T cells transiently
expressing HA-D 4.2 R and/or FLAG-D 4.4 R, by adding anti-FLAG antibody to the living cells.
Subsequently, cell lysates were made andmembrane-labeled receptors immunoprecipitated.
For immunoblotting, the same antibodies as in B were used. o Samples in which cells were
independently transfected and mixed posttransfection.
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