Figure 4.
ALS-FTLD mutant SQSTM1G427R of SQSTM1 displays impaired sequestration and clearance of ubiquitinated proteins. (A) MEFs sqstm1 KO stably expressing Flag-SQSTM1 WT or SQSTM1G427R mutant were treated with MG132, followed by immunofluorescent staining with anti-SQSTM1 antibody. Scale bar: 10 μm. (B) Quantification of the results in A was performed by counting the number of cells containing SQSTM1 inclusions. Then, the number of cells containing SQSTM1 inclusions was normalized to the total number of cells. Student t-test was used, and values are presented as the mean ± SEM (n = 3). *** p < 0.001. (C) MEFs sqstm1 KO stably expressing Flag-SQSTM1 WT and SQSTM1G427R mutant were treated with MG132, followed by cell fractionation. Soluble and insoluble fractions were subjected to immunoblot analysis with the indicated antibodies. (D) Quantification of the results in C was performed by normalized the level of insoluble SQSTM1 to the level of soluble SQSTM1. Student’s t-tests were used, and values are presented as the mean ± SEM (n = 4). ** p < 0.01. (E) Flag-SQSTM1 WT and SQSTM1G427R mutant stable cells were treated with MG132 (μM) for 16 h, as indicated, and then the medium was switched to serum starvation (S.S.). (F) Quantification of the results from E was obtained by normalizing the level of polyubiquitinated proteins to the level of ACTB, and further to SQSTM1-WT without S.S. Student-t test was used and values are presented as mean ± SEM (n = 3). * p < 0.05, ns, not significant.