Analysis of cytosolic and sarcomeric α-actinin during mitosis.

<p>(A) Synchronized NRVM using thymidine and Ro3306 reveal increased α-actinin in RIPA buffer cell extracts in association with an increase in mitotic index. NRVM were cultured on 12 mm glass cover slips and synchronized with thymidine and 9 μM Ro3306 as indicated in the timeline. Immunofluorescence staining with α-actinin antibody and DAPI were performed to determine the mitotic index of NRVM at the different time points. A separate synchronization experiment was also performed in parallel on cells cultured in 6-well plates. At the same time points cells were lysed in RIPA buffer to obtain total cellular protein and this was analyzed for α-actinin by Western blot. The average mitotic index data from three independent experiments is shown. (B) Cell cycle analysis based on Vybrant DyeCycle Violet DNA staining was performed on non-synchronized NRVM (top panel) and synchronized NRVM (bottom panel). The high DNA content population (G₂M population) increased in the cell cycle synchronized NRVM (4 hr after release from Ro3306) in comparison with non-synchronized NRVM. (C) Cytosolic and sarcomeric fractions of NRVM separated by FACS into different cell cycle stages based on DNA content were analyzed for α-actinin by Western blot analysis. (D) Phosphorylation status of cytosolic and sarcomeric α-actinin of FACS-sorted NRVM was determined using Phos-tag PAGE. GAPDH was used as a cytosolic marker. (E) Sarcomeric α-actinin phosphosites identified in large-scale phosphoproteomic screens. Upper bars show all sites reported in the <i>PhosphoSitePlus</i> database. The height of the bars corresponds to the number of species in which a given site was identified. Lower bars show phosphosites from mouse heart, with the height of the bar corresponding to the number of spectral matches, a semi-quantitative measure of phosphosite abundance. Residues of interest from the text are indicated.