Journal: Journal of Cell Science
Article Title: Lamin A/C phosphorylation at serine 22 is a conserved heat shock response to regulate nuclear adaptation during stress
doi: 10.1242/jcs.259788
Figure Lengend Snippet: Lamin A/C is phosphorylated at serine 22 upon HS. (A) Western blot (WB) analysis of lamin A/C (LAC), phospho-serine 22 lamin A/C (pSer22 LAC), phospho-serine 392 lamin A/C (pSer392 LAC) and heat shock factor 1 (HSF1) after 1–4 h heat shock (HS) at 42°C, and 3 h and 24 h recovery at 37°C in HeLa cells and primary human and mouse fibroblasts. Whole D. melanogaster flies were heat-shocked for 30 min at 37°C and left to recover for 3 or 7 h at 22°C. The average numerical values of signal intensities relative to loading control (GAPDH or actin) from individual experiments are shown below each blot. pSer22 and pSer392 lamin A/C levels were normalized to GAPDH and lamin A/C. (B) Quantification of pSer22 lamin A/C WB intensities upon HS and recovery in HeLa cells ( n =5), human fibroblasts, mouse fibroblasts and D. melanogaster fruit flies ( n =4). Plots show mean±s.d. and the individual datapoints. The shapes of the datapoints indicate each individual replicate. * P <0.05; ** P <0.01 (Mann–Whitney test). (C) Heat shock experiment with synchronized HeLa cells. Aphidicolin was used to synchronize cells to G1/S phase and nocodazole to synchronize cells to mitosis (positive control). GAPDH was used as a loading control ( n =2). (D) HeLa cells, human and mouse fibroblasts were cultured either in normal culture conditions or exposed to 4 h HS at 42°C, fixed and stained for lamin A/C (green), pSer22 lamin A/C (magenta) and DAPI (gray). The narrow panels show the orthogonal views of the yz -plane. Scale bars: 10 μm.
Article Snippet: The primary antibodies used were: mouse monoclonal anti-lamin A/C (1:10,000, 5G4, kindly provided by Prof. Robert D. Goldman, Northwestern University, IL), anti-lamin B1 (1:1000, ab16048, Abcam) rabbit polyclonal anti-heat shock factor 1 (1:1000, ADI-SPA-901, Enzo Life Sciences, Farmingdale, NY), rat monoclonal anti-heat shock factor 1 (1:1000, 10H8, StressMarq Biosciences, Victoria, Canada), rabbit monoclonal anti-phospho-lamin A/C serine 22 (1:1000, D2B2E, Cell Signaling Technology), rabbit polyclonal anti-phospho-lamin A/C serine 392 (1:1000, ab58528, Abcam), rabbit polyclonal anti-AKT (pan) (1:1000, C67E7, Cell Signaling Technology), rabbit monoclonal anti-pAKT T303 (1:1000, D6F8, Cell Signaling Technology), rabbit polyclonal anti-ERK1 (1:500; K-23, Santa Cruz Biotechnology), rabbit polyclonal anti-ERK2 (1:500, c-14, Santa Cruz Biotechnology), rabbit monoclonal anti-pERK1/2 (1:1000, D13.14.4E, Cell Signaling Technology), rabbit monoclonal anti-cleaved PARP-1 (1:1000, E-51, Abcam), rabbit monoclonal anti-caspase-3 (1:1000, 8G10, Cell Signaling Technology), rabbit monoclonal anti-cleaved caspase-3 (1:1000, Asp175, Cell Signaling Technology), rabbit monoclonal anti-Lap2α (1:5000, 245/2, kindly provided by Prof. Roland Foisner, University of Vienna), mouse monoclonal anti-HSP70/HSP72 (1:1000, C92F3A-5, Enzo Life Sciences), rat monoclonal anti-HSC70/HSP73 (1:1000, 1B5, Enzo Life Sciences), HRP-conjugated anti-GAPDH (1:5000, ab9385, Abcam) and mouse monoclonal anti-actin (1:1000, AC-40, Sigma-Aldrich).
Techniques: Western Blot, MANN-WHITNEY, Positive Control, Cell Culture, Staining
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