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. Author manuscript; available in PMC: 2021 Aug 24.
Published in final edited form as: Cell Rep. 2021 Jul 20;36(3):109399. doi: 10.1016/j.celrep.2021.109399

Figure 6. VCP-R155C is a hypomorphic allele and resembles VCP loss of function in mouse neurons.

Figure 6.

(A) Locomotor activity of VCP cRC mice and control VCPRC/FL mice at 6 and 10 months of age; paired t test between genotypes, **p < 0.01. Data represent mean ± SEM (error bars) (n = 10 mice/group).

(B) Time spent searching the quadrant where the hidden platform at both VCP cRC at 6 months (paired t test, *p < 0.05) and at 10 months (**p < 0.01) compared to control VCPRC/FL mice at each age. Data represent mean ± SEM (error bars) (n = 10 mice/group).

(C) LC3 (green), Lamp1 (red) immunofluorescence, and DAPI nuclear (blue) fluorescence of the cortex from 9-month-old control (C57), 9-month-old VCP-R155C heterozygous (VCPRC/FL), or VCP cRC (VCPRC/FL:CamkCre) mice at 3, 6 and 12 months of age. Scale bars, 30 μm.

(D) SQSTM1 (green), Lamp1 (red) immunofluorescence, and DAPI nuclear (blue) fluorescence of the cortex from 9-month-old control (C57), 9-month-old VCPR155C heterozygous (VCPRC/FL), or VCP cRC (VCPRC/FL:CamkCre) mice at 3, 6, and 12 months of age. Scale bars, 30 μm (n = 3 slices from four mice per group).

(E) Soluble and insoluble proteins from brain lysates of 9-month-old control (C57) or VCP cRC (VCPRC/FL:CamkCre) mice at 6, 9, and 12 months of age were immunoblotted with antibodies to VCP, Lamp2, TDP-43, SQSTM1, BAX, ubiquitin conjugates (FK2), GAPDH (loading control), and 14-3-3 (loading control).

(F) Immunoblot for BIP, PERK, CHOP, HSP70, and GAPDH (loading control) of brain lysates from 9-month-old control (C57) or VCP cRC (VCPRC/FL:CamkCre) mice at 6, 9, and 12 months of age.

(G) Quantitation band intensities of BIP, PERK, CHOP, HSP70, SQSTM1, and BAX relative to GAPDH (n = 4 animals per group). Quantification was determined by ImageJ software. Data represent mean ± SD (error bars) (data points represent mice). A one-way ANOVA was used for statistical testing, treating mice brains as an independent sample; for BIP is F(3,12) = 10.32, p < 0.0012; for p-PERK is F(3,12) = 6, p = 0.0062; for CHOP is F(3,12) = 19.19, p = 0.0001; for HSP70 is F(3,12) = 17.19, p = 0.0001; for SQSTM1 is F(3,12) = 5, p = 0.0127; for BAX is F(3,12) = 2.297, p = 0.1296. Post hoc comparisons using a Bonferroni test, *p < 0.05.