Next, to identify the essential region of Plk2 required for the interaction with NPM/B23, we generated Flag-tagged Plk2 truncated versions (Fig. 2E) that were ectopically expressed in 293T cells, and immunoprecipitated with Flag-antibodies.
To examine the function of each phosphorylation site, and to confirm the specificity of the phospho-specific antibodies, we generated Flag-tagged menin mutants with non-phosphorylatable Serine to Alanine substitutions at each site.
To determine whether mutant PINK1 affects mitochondrial function, we generated Flag-tagged wild type, L347P, E417G or Del 245 PINK1-adenovirus to infect human neuroblastoma SH-SY5Y cells with high efficiency.
We generated FLAG-tagged protein as described in the Materials and Methods.
The generated Flag-tagged mutants were subcloned in both pMK33 and pCasperhs83 vectors as previously described [ 35].
We generated FLAG-tagged constructs of Eif1al6 (FLAG- Eif1al6) and Eif1al9 (FLAG- Eif1al9) and transfected them into ES cells, followed by the analysis of the nascent protein synthesis determined via AHA labelling and immunofluorescence.
As our current study shows the involvement of HSP90 in Tax-mediated HMW-IKK formandon and NF-κB activation, we generated Flag-tagged serial deletion mutants of HSP90 and CDC37 to examine their potential effects on Tax activity.
For mammalian cell expression, we generated FLAG-tagged XBP1u with its 3′ UTR by following the previously described procedure (Yanagitani et al., 2009) and cloned into pcDNA5/FRT/TO (Invitrogen, Carlsbad, CA).
To understand the relative distribution of mtHsp70 in soluble and insoluble fractions in mammalian cells, we generated FLAG-tagged mammalian constructs of wild-type and human mtHsp70 PD variants.
To investigate the inhibitory function of FOXG1 on TGF- β-induced p21WAF1/CIP1 expression and cell proliferation, we first generated Flag-tagged FOXG1 stable-expressing clones from two ovarian cancer cell lines, SKOV3 and A2780cp.
Based on our previous experiments defining Ser408, Thr404 and Thr400 as amino acids targeted by CK2 [ 31], we here generated FLAG-tagged triple A and triple E phospho-site mutated occludin constructs to mimic unphosphorylated and CK2-phosphorylated occludin.
