Investigating Protein Interactions via WB and Co-IP

After the cells were subjected gene transfection or drug treatments, cell lysates were prepared and quantified according to a previous study [14 (link)]. The western blot was carried out as described previously [13 (link)], while the co-immunoprecipitation kit (Cat. #26419) from Thermo Scientific (Waltham, MA, USA) was used according to the manufacturer’s instructions with the following antibodies: anti-ROC1 (Abcam, Cambridge, MA, USA); anti-cyclin D1, anti-Cdc25c, anti-SUFU, anti-Gli1, anti-GAPDH, and anti-HA (Abcam, Hangzhou, China); and anti-Gli2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

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Wang W., Qiu J., Qu P., Chen H., Lan J., Chen H., Li L, & Gu M. (2021). Regulator of cullins-1 (ROC1) negatively regulates the Gli2 regulator SUFU to activate the hedgehog pathway in bladder cancer. Cancer Cell International, 21, 75.

Publication 2021

anti-ROC1 anti-cyclin D1 anti-Cdc25c anti-SUFU anti-Gli1 anti-GAPDH anti-HA anti-Gli2

Antibodies Cdc25c Cell Co immunoprecipitation Cyclin d1 Drug Gapdh Gene Transfection Western blot

Corresponding Organization :

Other organizations : Nanjing Medical University, Jiangsu Province Hospital, Shanghai First People's Hospital, Shanghai Jiao Tong University, Nantong University, Xuzhou Medical College, Yancheng First People's Hospital

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Variable analysis

independent variables

Gene transfection
Drug treatments

dependent variables

Protein levels (detected by Western blot)
Protein-protein interactions (detected by co-immunoprecipitation)

control variables

Cell lysates were prepared and quantified according to a previous study [14]
Western blot was carried out as described previously [13]
Co-immunoprecipitation was performed using a kit from Thermo Scientific according to the manufacturer's instructions

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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