Anti flag tag monoclonal antibody

Co-IP assay was used to identify the interactions of TA05575 and the prey proteins in HEK293T cells. The cells were cultured in 10-cm cell culture dishes (Thermo Fisher Scientific, MA, USA) at an initial density of 2 × 10⁶ cells per dish. Fourteen hours later, 10 µg of TA05575 plasmid and 10 µg of the prey plasmids were co-transfected into the cells. After 48 h of transfection, the cells were washed two times with cold PBS and lysed with 600 μl of IP/lysis buffer (Thermo Fisher Scientific, MA, USA) containing the protease inhibitor (Roche, Basel, Switzerland, #4693132001) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland, #4906845001) on ice. Then, the supernatants of cell lysate were obtained by centrifuging at 16,000 × g at 4°C for 10 min. The immunoprecipitation experiment was carried out with an anti-Flag tag monoclonal antibody derived from mouse (Sigma, #F1804) using a Pierce™ Co-Immunoprecipitation kit (Thermo Fisher Scientific, MA, USA, #26149). In addition, p3×Flag-CMV and pcDNA3.1 plasmid served as negative controls, were also co-transfected into HEK293T cells. The elution from the Co-IP experiment was analyzed with western blotting. anti-Flag tag monoclonal antibody (Sigma, #F1804) and anti-Myc tag monoclonal antibody derived from rabbit (CST, #2278S) were used to identify its target protein, respectively.

Anti-Cullin1 and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Calbiochem (La Jolla, CA). Anti-EF1α, anti-Skp1, and anti-ubiquitin antibodies were obtained from Millipore (Billerica, MA). The Anti-flag tag monoclonal antibody was bought from Sigma chemicals (St. Louis, MO). HRP-conjugated anti-mouse and anti-rabbit IgG antibodies for immunoblot assays were obtained from Santa Cruz Biotech Inc. (Santa Cruz, CA). AlexaFluor488- and AlexaFluor594-conjuagted anti-mouse and anti-rabbit IgG antibodies were purchased from Molecular Probes (Invitrogen) for immunofluorescence assays. Human pooled sera were prepared from 10 scrub typhus patients (IFA titer≥1∶1280) and 10 healthy volunteers following institutional review board approval and the receipt of informed consent from all subjects.