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. 2020 Nov 1;31(23):2522–2536. doi: 10.1091/mbc.E20-05-0290

FIGURE 5:

FIGURE 5:

Phosphorylated ALS-mutant FUS is present in cytoplasmic granules. (A) H4 cell transfected with GFP-FUS(R495X) for 24 h or untransfected control (*). GFP-FUS(R495X) was pulled down from cell lysates using GFP immunoprecipitation (IP). IP products were analyzed by Western blot and probed with anti-FUS (Santa Cruz) and phospho-FUS (pSer26, pSer30, pThr71, and pSer96). GFP-FUS(495X) is denoted with an arrow at roughly 100 kDa. (B) Ectopic expression of mutant FUS(R495X) in H4 cells with N- or C-terminal GFP. Anti-phospho-FUS(pSer30) antibody was used to probe for phosphorylated FUS. (C) Quantification of number of cells expressing diffuse, granular, or aggregated FUS(R495X) at 6, 8, or 24 h posttransfection (n = 3). (D) Quantification using Pearson’s coefficient of correlation of phospho-FUS (pSer26, pSer30, pSer57, pThr71, and pSer96) to the GFP-FUS(R495X) signal; error bars represent 95% CI (n = 30). (E) H4 cells transfected with GFP-FUS(495X) treated with torin 2, 6 h posttransfection. Cells were analyzed 8 h posttransfection and probed with phospho-FUS antibodies. Error bars represent 95% CI (n = 30).