Immunoprecipitation and Western Blotting of C3aR and CD11a in Activated NK Cells

NK cells were isolated and activated with IL2 for 96 hours as described above. The cells were collected and washed with ice cold PBS at 4^oC (1500 rpm for 10 minutes). A standard immunoprecipitation protocol was followed as described in a previous publication (25 (link)). 1 μg of anti-human C3aR (Cat# SC-133172, Santa Cruz Biotechnology) and 1 μg of anti-human CD11a (Cat# 350602, Biolegend) were used in 1 mg of total protein for pulldown. Pulled down eluates were processed for Western blotting as described below.
For mouse NK-cell immunoprecipitation, mouse NK cells were treated with IL2 for 96 hours as described. The activated mouse NK cells were processed as described above. Pulldown assays were performed for C3aR using anti-C3aR ((Cat# SC-133172, Santa Cruz Biotechnology). Polyclonal anti CD11a polyclonal antibody (Cat# BS 20370R, Bioss) and Anti C3aR polyclonal antibody (Cat# BS 2955R, Bioss) were used for immunoblotting.

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Nandagopal S., Li C.G., Xu Y., Sodji Q.H., Graves E.E, & Giaccia A.J. (2021). C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models. Cancer immunology research, 10(2), 245-258.

Publication 2021

anti-human CD11a anti-C3aR

Anti antibody Anti cd11a Assays C3ar Cells Cold Human Immunoprecipitation Mouse Nk cells Protein

Corresponding Organization : University of Oxford

Other organizations : La Jolla Bioengineering Institute

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Variable analysis

independent variables

IL2 treatment duration (96 hours)

dependent variables

Protein interactions/associations (C3aR and CD11a)

control variables

Isolation and activation of NK cells
Immunoprecipitation protocol
Antibodies used for pulldown (anti-human C3aR, anti-human CD11a, anti-mouse C3aR)
Antibodies used for immunoblotting (anti CD11a, anti C3aR)

controls

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

Annotations

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