[14]). cell nucleus. Mass spectrometry discovered hundreds of proteins, including 12 potentially novel PD 169316 PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)Ps conversation partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of proteinClipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation. were performed at a 120K resolution (at 200 em m /em / em z /em ) with a 5 105 ion count target. Tandem MS was performed by isolation at 1,5 Th with the quadrupole; HCD fragmentation, with a normalised PD 169316 collision energy of 30; and rapid scan MS analysis, in the ion trap. The MS/MS ion count PEBP2A2 target was set to 104, and the maximum injection time was 35 ms. Only those precursors with a charge state of 2C6 were sampled for MS/MS. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top velocity mode with 2 s cycles [31]. 2.5.3. Data Analysis All data were analysed and quantified with the MaxQuant software version 1.6.2.1 [32]. The false discovery rate (FDR) was set to 1% for both proteins and peptides, and we specified a minimum length of seven amino acids. The Andromeda search engine was used for the MS/MS spectra search PD 169316 against the Human database (downloaded from Uniprot on September 2017, made up of 20,142 entries). The enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. The dithiomethylation of cysteine was selected as a fixed modification, and N-terminal protein acetylation and methionine oxidation, as variable modifications. The match between runs feature of MaxQuant was used to transfer identifications to other LC-MS/MS runs based on their masses and retention occasions (maximum deviation, 0.7 min), and this was also used in quantification experiments. Quantifications were performed with the label-free algorithms described recently. Data analysis was performed using the Perseus 1.6.1.3 software. 2.6. Indirect Immunofluorescence Microscopy Cells produced on glass coverslips were washed with PBS three times, then fixed and permeabilised simultaneously with 0.1% Triton X-100 and PD 169316 4% formaldehyde in PBS for 10 min at room temperature (RT). The samples were blocked with 5% normal goat serum in PBS PD 169316 for 1 h at RT, incubated with primary antibodies diluted in PBS for 1 h at RT and then washed with PBS. The samples were then incubated with appropriate secondary antibodies diluted in PBS for 1 h at RT, washed with PBS and mounted in 90% glycerol and 1% 1,4-diazabicyclo-octane (DABCO). Images were acquired using a super-resolution Leica TCS SP8 STED 3X microscope with a 100 (NA = 1.4) oil immersion objective and the LAS X software version 3.5.5. The acquired images were deconvolved using the Huygens Professional software. The graphs in Physique 1c were made using the FiJi software with a custom-made macro using em Analyze ? Plot Profile /em , which shows pixel intensities along a line selection in RGB images. Open in a separate window Physique 1 Detection of phosphatidylinositol 4-phosphate (PI(4)P) at the nuclear envelope and in the nucleoli. Super-resolution STED microscopy revealed the localisation of PI(4)P in the nuclear lamina, the nucleoli, nuclear speckles and nucleoplasmic foci. (a) Fluorescently labelled PI(4)P and lamin B1. (b) PI(4)P co-localises with lamin B1. (c) Intensities of pixels along the lines in b, PI(4)P (green) and lamin B1 (purple). Lines are drawn in a direction from the nucleoplasm to the cytoplasm (from bottom to top). Graphs were made using the FiJi software. (d) Localisation of PI(4)P and a protein C23, a marker of the nucleoli. (e) PI(4)P localises to the nucleoli, but the intensity of.
