Anti flag monoclonal antibody

For the co-immunoprecipitation (co-IP) assays, A549 cells were transfected with Flag-tagged RPS27a or vector control, then lysed. Subsequently, 70% of the lysate was incubated with anti-Flag monoclonal antibody (Cell signaling technology, USA) or control IgG, and the remaining 30% of the lysate was analyzed with IB. In vitro ubiquitination experiments followed protocols from previous studies using the Ni²⁺-NTA purification method. The A549 cells were transfected with His-Ub plasmids after transfection of RPS27a-siRNA for 24 h, then treated with 40 μM MG132 for 6 h. Subsequently, 70% of the lysate was incubated anti-His monoclonal antibody (Cell signaling technology) and used for ubiquitination experiments with co-IP assays; the bead-bound proteins and the other 30% of the lysate were analyzed with IB [29 (link)].

For in vivo PGAM5 ubiquitylation assay, HEK293T and HL-1 cells were transfected with Myc-MARCH2, HA-PGAM5, and Flag-ubiquitin using Lipofectamine 3000. 24 h later, whole cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, with phosphatase and protease inhibitors). The lysates were centrifuged to obtain supernatant proteins and incubated with anti-Flag antibody (Cell Signaling Technology) for 3 h and protein A/G agarose beads (Santa Cruz Biotechnology) for 3 h at 4 °C. Then the immune complex was washed extensively for four times with lysis buffer and boiled with SDS-PAGE sample buffer for 10 min. Ubiquitination was analyzed by immunoblotting with anti-Flag monoclonal antibody (Cell Signaling Technology).