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. 2021 Mar 22;10:e61590. doi: 10.7554/eLife.61590

Figure 2. Mouse model of the human SWIPP1019R mutation displays decreases in WASH complex components.

(A) Mouse model of the human SWIPP1019R missense mutation created using CRISPR. A C>G point mutation was introduced into exon29 of murine Washc4, leading to a P1019R amino acid substitution. We hypothesize (H1) that this mutation causes instability of the WASH complex. (B) Representative western blot and quantification of WASH components, Strumpellin and WASH1 (predicted sizes in kDa: 134 and 72, respectively), as well as loading control β-tubulin (55 kDa) from adult whole-brain lysate prepared from SWIP WT (Washc4C/C) and SWIP homozygous MUT (Washc4G/G) mice. Bar plots show quantification of band intensities normalized to β-tubulin, expressed as a % of WT (n=3 mice per genotype). Strumpellin (WT 100.0±5.1%, MUT 3.8±0.9%, t2.14=18.60, p=0.0021) and WASH1 (WT 100.0±4.3%, MUT 1.1±0.4%, t2.1=22.77, p=0.0018) were significantly decreased. Equivalent amounts of protein were analyzed in each condition (β-tubulin: WT 100.0±8.2%, MUT 94.1±4.1%, U=4, p>0.99). (C) Schematic of experimental techniques used to interrogate the effect of the SWIPP1019R mutation in subsequent figures: spatial proteomics (top), confocal and electron microscopy (middle), and mouse behavioral tasks (bottom).

Figure 2.

Figure 2—figure supplement 1. Overexpression of SWIPP1019R decreases WASH complex binding in cultured cells.

Figure 2—figure supplement 1.

(A) Schematic showing overexpression of WT or MUT SWIPP1019R in HEK293T cells followed by immunoprecipitation. (B) Western blots of input (5%, left) and immunoprecipitated (IP, right) protein. Two samples per condition were run on two separate gels, n=4 separate experiments. (C) Quantification of B normalized to WT. Strumpellin (WT 100.0±6.8%, MUT 54.8±8.0%, t5.9=4.290, p=0.0054), WASH1 (WT 100.0±7.3%, MUT 41.4±4.4%, t4.9=6.902, p=0.0011), HA (WT 100.0±4.1%, MUT 107.8±4.1%, t6.0=1.344, p=0.2275). Data reported as mean ± SEM, error bars are SEM. **p<0.01, two-tailed t-tests.