Experiments*. (DOC)IFN-c ELISPOT AssayIFN-c ELISPOT assays were performed as previously

Experiments*. (DOC)IFN-c ELISPOT AssayIFN-c ELISPOT assays were performed as previously FCCP described [23]. Briefly, wells of 96-well-plates with nitrocellulose membrane inserts were incubated with a capture anti-IFN-c mAb (clone 1D1K, Mabtech, Nacka, Sweden) for 24 h at 4uC. Plates were washed, and the CTL (16105) generated in IVS assays as described above were added to each well followed by mDC (2.56104) pulsed with a tumor cell lysate. Plates were then incubated for 24 h at 37uC. Next, cells were removed by extensive washing, and a biotinylated secondary anti-IFN-c Ab (clone 7-B61, Mabtech) was added for 2 h. After washing, plates were incubated with the avidin-peroxidase complex reagent, and aminoethylcabazole was added as a substrate. The reaction was terminated after 5 min, and spots were counted by computer-Author ContributionsConceived and designed the experiments: TLW SF JE. Performed the experiments: BS MH PS. Analyzed the data: BS TLW. Contributed reagents/materials/analysis tools: BS JE SF. Wrote the paper: BS TLW.
Microtubules are cytoskeletal filaments that play important roles in the organization, shape, motility and division of eukaryotic cells [1]. Microtubules consist of ab-tubulin heterodimers that selfassemble head-to-tail to form protofilaments and laterally to form a hollow tube. The ab-tubulin subunits can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) including acetylation, polyglutamylation, polyglycylation, detyrosination, phosphorylation and palmitoylation that are thought to regulate the polymerization properties of tubulins and/or their interactions with microtubule associated proteins (MAPs) and motor proteins. Thus, PTMs provide functional specialization to microtubules ranging from Indolactam V site structural support to intracellular trafficking [2]. A prominent PTM of microtubules is the acetylation of the eamino group of Lysine-40 (K40) of a-tubulin [3,4]. K40 acetylation has been widely noted due to the availability of a monoclonal antibody 6-11B-1 that binds to K40-acetylated atubulin across a wide variety of species [5]. K40 acetylation accumulates on a subset of cytoplasmic microtubules as well as microtubules in the spindle, axon and cilia. Despite its widespread occurrence, the functional significance of K40 acetylation remains unclear. Microtubule acetylation has been implicated in regulating a variety of cellular functions including ciliary assembly, intracellular trafficking, cell motility, and axon outgrowth [2,6]. These effects may be due to direct effects of K40 acetylation on microtubule dynamics as acetylation is generally believed to mark“stable” microtubules (resistant to depolymerizing conditions), yet whether K40 acetylation directly influences microtubule dynamics is controversial [7?1]. 1527786 K40 acetylation can influence interactions between neighboring ab- tubulin subunits and thus affect protofilament number and organization in worms [12,13]. Notably, K40 acetylation has been suggested to directly impact events on the surface of cellular microtubules such as severing [14] and the binding and motility of kinesin-1 and cytoplasmic dynein motors [15?8]. The K40 residue resides in a loop of a-tubulin that was found disordered in both the electron crystallographic structure of abtubulin and a high resolution cryo-EM microtubule reconstruction, but is thought to be positioned in the lumen of the microtubule [19?1]. How a luminal residue becomes modified by cytoplasmic enzy.Experiments*. (DOC)IFN-c ELISPOT AssayIFN-c ELISPOT assays were performed as previously described [23]. Briefly, wells of 96-well-plates with nitrocellulose membrane inserts were incubated with a capture anti-IFN-c mAb (clone 1D1K, Mabtech, Nacka, Sweden) for 24 h at 4uC. Plates were washed, and the CTL (16105) generated in IVS assays as described above were added to each well followed by mDC (2.56104) pulsed with a tumor cell lysate. Plates were then incubated for 24 h at 37uC. Next, cells were removed by extensive washing, and a biotinylated secondary anti-IFN-c Ab (clone 7-B61, Mabtech) was added for 2 h. After washing, plates were incubated with the avidin-peroxidase complex reagent, and aminoethylcabazole was added as a substrate. The reaction was terminated after 5 min, and spots were counted by computer-Author ContributionsConceived and designed the experiments: TLW SF JE. Performed the experiments: BS MH PS. Analyzed the data: BS TLW. Contributed reagents/materials/analysis tools: BS JE SF. Wrote the paper: BS TLW.
Microtubules are cytoskeletal filaments that play important roles in the organization, shape, motility and division of eukaryotic cells [1]. Microtubules consist of ab-tubulin heterodimers that selfassemble head-to-tail to form protofilaments and laterally to form a hollow tube. The ab-tubulin subunits can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) including acetylation, polyglutamylation, polyglycylation, detyrosination, phosphorylation and palmitoylation that are thought to regulate the polymerization properties of tubulins and/or their interactions with microtubule associated proteins (MAPs) and motor proteins. Thus, PTMs provide functional specialization to microtubules ranging from structural support to intracellular trafficking [2]. A prominent PTM of microtubules is the acetylation of the eamino group of Lysine-40 (K40) of a-tubulin [3,4]. K40 acetylation has been widely noted due to the availability of a monoclonal antibody 6-11B-1 that binds to K40-acetylated atubulin across a wide variety of species [5]. K40 acetylation accumulates on a subset of cytoplasmic microtubules as well as microtubules in the spindle, axon and cilia. Despite its widespread occurrence, the functional significance of K40 acetylation remains unclear. Microtubule acetylation has been implicated in regulating a variety of cellular functions including ciliary assembly, intracellular trafficking, cell motility, and axon outgrowth [2,6]. These effects may be due to direct effects of K40 acetylation on microtubule dynamics as acetylation is generally believed to mark“stable” microtubules (resistant to depolymerizing conditions), yet whether K40 acetylation directly influences microtubule dynamics is controversial [7?1]. 1527786 K40 acetylation can influence interactions between neighboring ab- tubulin subunits and thus affect protofilament number and organization in worms [12,13]. Notably, K40 acetylation has been suggested to directly impact events on the surface of cellular microtubules such as severing [14] and the binding and motility of kinesin-1 and cytoplasmic dynein motors [15?8]. The K40 residue resides in a loop of a-tubulin that was found disordered in both the electron crystallographic structure of abtubulin and a high resolution cryo-EM microtubule reconstruction, but is thought to be positioned in the lumen of the microtubule [19?1]. How a luminal residue becomes modified by cytoplasmic enzy.