Probing ER-TRIM3 Interactions via Immunoprecipitation

Immunoprecipitation was performed as described in a previous study [20 (link)]. MCF-7 total cell lysates were precleared with rabbit IgG for 2 h and subsequently immunoprecipitated with an anti-ER alpha antibody (SC8005, Santa Cruz, 1:200) overnight, while rabbit IgG (Santa Cruz, 1:200) was used as the negative control. The bound proteins were analyzed by immunoblotting with an anti-TRIM3 antibody (HAP043396, Sigma, 1:2000). For the overexpression experiment, HEK293 cells were transfected with 5 μg of GFP-TRIM3 (full-length or domain deletion mutants) and ER alpha plasmid (full-length or domain deletion mutants) in 10 cm dishes. Cell lysates were precleared with IgG and subsequently incubated with an anti-GFP (ab290, Abcam, 1:200) antibody, while rabbit IgG was used as the negative control. The bound proteins were analyzed by western blotting.

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Zhuang T., Wang B., Tan X., Wu L., Li X., Li Z., Cai Y., Fan R., Yang X., Zhang C., Xia Y., Niu Z., Liu B., Cao Q., Ding Y., Zhou Z., Huang Q, & Yang H. (2022). TRIM3 facilitates estrogen signaling and modulates breast cancer cell progression. Cell Communication and Signaling : CCS, 20, 45.

Publication 2022

rabbit IgG SC8005 HAP043396 ab290

Anti antibody Antibody Cell Deletion Dishes Er alpha Hek293 cells Immunoprecipitation Mcf 7 cell Plasmid Proteins Rabbit

Corresponding Organization : Huazhong Agricultural University

Other organizations : Xinxiang Medical University, Wenzhou Medical University, Dongyang People's Hospital, Karolinska Institutet, Second Hospital of Shandong University

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Variable analysis

independent variables

Expression of GFP-TRIM3 (full-length or domain deletion mutants)
Expression of ER alpha plasmid (full-length or domain deletion mutants)

dependent variables

Interaction between TRIM3 and ER alpha proteins

control variables

MCF-7 total cell lysates
Rabbit IgG (used for preclearing and as negative control)

controls

Positive control: Anti-ER alpha antibody (SC8005, Santa Cruz, 1:200) used for immunoprecipitation
Negative control: Rabbit IgG (Santa Cruz, 1:200) used for immunoprecipitation
Positive control: Anti-GFP antibody (ab290, Abcam, 1:200) used for immunoprecipitation
Negative control: Rabbit IgG used for immunoprecipitation

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