Evaluation of the Strep-tag recombinant proteins expression and Western blot analysis of NBN, p26 and p70 interactors.

<p>(<b>A</b>) Protein lysates, obtained after 24 and 48 h from HEK293 transient tranfections, were analyzed by Western blot using a StrepMAB-Classic horse radish peroxidase conjugated antibody. The highest level of expression was observed after 48 h from transfection, for all the recombinant proteins. Protein extracts obtained from HEK293 cells transfected with the empty vector were used as negative control. (<b>B</b>) Check of the expression of the Strep-tag recombinant proteins. Immunoblots were performed using the protein eluates obtained from HEK293 transfected cells either untreated or exposed to 2 Gy of X-rays, lysed after 30 minutes and purified by Strep-tag chromatography. Filters were probed with the anti-NBN antibody directed against the full-length protein. (<b>C</b>) Protein eluates obtained from HEK293 transfected cells exposed to 2 Gy of X-rays were lysed after 30 minutes, purified by Strep-tag chromatography, and analysed by Western blot using the following antibodies: anti-53BP1 rabbit polyclonal, anti-ATM mouse monoclonal, anti-ATM pSer1981 mouse monoclonal, anti-BRCA1 mouse monoclonal, anti-CHK2 pThr68 rabbit polyclonal, anti-CHK2 mouse monoclonal, anti-CtIP rabbit polyclonal, γ-H2AX rabbit polyclonal, anti-Hsp90 rabbit polyclonal, anti-MRE11 mouse monoclonal, anti-NBN mouse monoclonal, anti-PARP1 mouse monoclonal, anti-PP2A rabbit polyclonal, anti-PML rabbit polyclonal, anti-SMC1 rabbit polyclonal.