Fig. 4: The Y65C mutation significantly reduces PQBP1 protein levels. | Nature Communications

Fig. 4: The Y65C mutation significantly reduces PQBP1 protein levels.

From: The missense mutation Y65C in PQBP1 causes microcephaly and cognitive deficits through a combination of partial loss-of-function and gain-of-function effects

Fig. 4: The Y65C mutation significantly reduces PQBP1 protein levels.

a Immunofluorescence staining shows the PQBP1 expression in the cortex at E12.5. Scale bar, 200 μm. Shown are representative results from three independent experiments. b Western blots using anti-PQBP1FL antibodies to detect PQBP1 in P30 WT and Pqbp1Y65C/Y cortices and quantification of the relative PQBP1 level. n = 3 mice in each group, p = 0.0054. c Quantification of Pqbp1 mRNA levels in P30 WT and Pqbp1Y65C/Y cortices. n = 5 mice in each group, p = 0.8461. d Overexpression of Flag-PQBP1 or Flag-PQBP1Y65C protein in SK-N-BE(2) cells. Flag-PQBP1, black line arrows; endogenous PQBP1, black solid arrows, respectively. The right panel is a quantification of the relative Flag-PQBP1 level. n = 4 biologically independent experiments in each group, 12 h: p = 0.3383; 18 h: p = 0.0746; 24 h: p < 0.0001; 36 h: p = 0.0006; 48 h: p = 0.0027; 72 h: p = 0.0096. e Overexpression of Flag-PQBP1 or Flag-PQBP1Y65C protein in 293 T cells for 36 h, 100 μg/ml CHX was added. Flag-PQBP1, black line arrows; endogenous PQBP1, black solid arrows, respectively. The right panel is a quantification of the relative Flag-PQBP1 level. n = 4 biologically independent experiments in each group, 3 h: p = 0.1242; 6 h: p = 0.0703; 9 h: p = 0.0016; 12 h: p = 0.0002. f Overexpression of Flag-PQBP1Y65C in 293 T cells for 36 h, 100 μg/ml CHX, and 10 μM MG132 were added. Flag-PQBP1, black line arrows; endogenous PQBP1, black solid arrows, respectively. The right panel is a quantification of the relative Flag-PQBP1Y65C level. n = 3 biologically independent experiments, 3 h: p = 0.3564; 6 h: p = 0.0765; 9 h: p = 0.0073; 12 h: p = 0.0003. g Overexpression of WT and mutant PQBP1 protein in SK-N-BE(2) cells for 36 h. Flag-PQBP1, black line arrows; endogenous PQBP1, black solid arrows, respectively. The panel below is a quantification of the relative Flag-PQBP1 level. n = 4 biologically independent experiments, WT vs. Y65C (TGC): p < 0.0001; Y65C (TGT) vs. Y65C (TGC): p = 0.1701; Y65F vs. Y65C (TGC): p = 0.0049; Y65S vs. Y65C (TGC): p = 0.0001; Y65W vs. Y65C (TGC): p = 0.0041; Y65C,C60S vs. Y65C (TGC): p = 0.0004. All quantification data are represented as mean ± SD. b–f, g two-tailed unpaired Student’s t test. ns>0.05, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Source data are provided as a Source Data file.

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