Fig 1: BYHW, NXT, and YYTN can reverse the expression of key genes. (a–g) The proteins levels of Rpl18, Eif3c, Rhoc, Gng13, and Gng10. Representative images of WB experiments are shown. Data are expressed as the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, and n = 3/group.
Fig 2: Validation of proteins expressed in pooled fibroblast samples of healthy controls, and in fibroblasts from patients with Parkinson's disease carrying GBA and PARK2 variants. (A) Representative western blots of ANXA2, RPL18 and COL1A1 in the GBA group. (B) Representative western blots of TUBB, COL1A1 and ANXA2 in the PARK2 group. ACTB was used for normalization of protein loading in each sample. (C and D) Relative ratios of protein expression levels obtained from western blot and label-free quantitative proteomic analyses. All protein levels were normalized to those of the pooled healthy control fibroblasts. Data are presented as the mean ± standard deviation of at least two independent experiments. *P<0.05, **P<0.01 vs. control. PD, Parkinson's disease; GBA, acid-β glucosidase; PARK2, parkin; WB, western blot; label-free, label free quantitative proteomics.
Fig 3: Candidate proteins identified with BONLAC screen of de novo proteome exhibit altered expression levels in APP/PS1 mice.a Western blot quantitation of candidate proteins selected from BONLAC screen in 3–5-month-old wild-type (WT; light bars) and APP/PS1 (dark bars) mice, normalized in- lane to total protein (as assessed by MemCode or Ponceau stain [note only a portion of the entire analyzed lane is presented in the figure]) and expressed relative to age-matched WT (Number of biologically independent animals samples: APP 3–5 month n = 8, 12+ month n = 6; EAAT1 n = 3; Gap-43 n = 6; Hsp1a1 n = 3; Rpl13 3–5 month n = 9, 12+ month n = 12; Rpl18: n = 7); representative western blots show selected protein levels in WT and APP/PS1 mouse hippocampal lysates. b Representative western blot and quantitation of candidate proteins selected from BONLAC screen in 12+-month-old WT and APP/PS1 mice. c Comparison table showing average fold change in de novo synthesis of candidate proteins in young and aged APP/PS1 vs WT littermates as identified by BONLAC (young n = 5 biologically independent samples; aged n = 7 biologically independent samples) compared to change in total expression quantified by western blot. Statistical significance calculated between APP/PS1 and WT samples using unpaired two-tailed t-tests; young APP: t = 3.378, 95% confidence interval = 0.4571–2.047, effect size = 1.252 ± 0.3707, df = 14, p = 0.0045; young EAAT1: t = 2.945, 95% confidence interval = 0.0146–0.4966, effect size = 0.2556 ± 0.0868, df = 4, p = 0.0422; aged APP: t = 13.95, 95% confidence interval = 1.027–1.424, effect size = 1.226 ± 0.08782, df = 9, p < 0.0001; aged GAP-43: t = 2.266, 95% confidence interval = −0.9047 to −0.01774, effect size = −0.4612 ± 0.2035, df = 12, p = 0.0427. d Linear regression showing correlation between average de novo synthesized protein fold change (as determined by BONLAC) vs. average total protein fold change (as determined via western blot) of candidate proteins in the APP/PS1 vs. WT hippocampus at 3–5 months (white circles) and 12+ months of age (teal circles; r 2 value = 0.690, vertical error bars: BONLAC; horizontal error bars: western blot; p = 0.0207). Graphs in a, b and d show mean ± SEM.
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