Figure 1.
Variant identification and amino acid sequence alignment.
(A) Chromatograms of direct cDNA sequencing of Y52C and M115I. (B) Restriction analysis of Y52C and M115I. On agarose gel, Y52C creates new PstI restriction site by mismatch PCR and leads to an additional 222 bp band, whereas M115I loses restriction by PaeI and leads to 479 bp band. (C) Evolutionary conservation of the regions of FBXO7 Y52C and M115I using the program Vector NTI.
Table 1.
Primers and conditions for PCR amplification of FBXO7 cDNA and genomic DNA.
Table 2.
Genotype and allele distributions and association analysis.
Figure 2.
Expression of FBXO7 protein in HEK-293T cells.
(A) Confocal microscopy examination of Tyr52 and Cys52 FBXO7-EGFP protein in HEK-293T cells. HEK-293T cells were transfected with Tyr52 and Cys52 FBXO7-EGFP for two days and stained with DAPI to detect nuclei. The green (FBXO7 fusion protein) to blue (nuclei staining) fluorescence signal was quantified and shown below. (B) Western blot analysis of pEGFP-N1 vector (Vec) and Tyr52 (WT) and Cys52 FBXO7-EGFP transfected cells using FBXO7 antibody and anti-tubulin and anti-neomycin antibodies as loading and transfection controls, respectively. (C) Tyr52 and Cys52 FBXO7-EGFP proteins in transfected cells were analyzed after blocking of new protein synthesis in a cycloheximide (200 µg/ml) chase experiment (cells were harvested after 6, 12, 24, 36, and 48 hr). Data are represented as the means ± S.D. of three separate experiments.
Figure 3.
The homology models of Tyr52 (WT) FBXO7 and Cys52 (Y52C).
Protein models were shown in secondary representation. Tyr52 and Cys52 residue were shown in amino acid structures. The hydrogen bond interaction was presented in yellow dash line.
Figure 4.
Western blot analysis of NF-κB signaling pathway protein TRAF2.
HEK-293T cells were tranfected with Tyr52 or Cys52 FBXO7 construct and harvested after two days. Transfected FBXO7-EGFP or endogenous TRAF2 protein was detected using specific antibodies and anti-actin and anti-neomycin antibodies as the loading and the transfection control, respectively. Data are represented as the means ± S.D. of three separate experiments.
Figure 5.
Co-immunoprecipitation of Tyr52 or Cys52 FBXO7-EGFP protein and ubiquitination of TRAF2.
(A) HEK-293T cells were transiently transfected with Tyr52 or Cys52 FBXO7-EGFP construct. After 48 h, cell lysates were prepared (Input, left panel) and immunoprecipitations (IP, right panel) were performed with anti-TRAF2 antibody. Normal IgG was used as a negative control for IP. Cell lysates and immunoprecipitates were analyzed with anti-EGFP, anti-TRAF2, anti-ubiquitin or anti-actin antibody. (B) Quantification of immunoprecipitated FBXO7-EGFP, TRAF2 and ubiquitin in HEK-293T cells transiently transfected with Tyr52 or Cys52 FBXO7-EGFP construct for 2 days. Data are represented as the means ± S.D. of three separate experiments.
Figure 6.
Toxic effects of MPP+ in FBXO7 suppressed or over-expressed cells.
(A) HEK-293T and SH-SY5Y cells were transfected with FBXO7-specific or control siRNA. After 48 h, cell lysates were prepared and Western blot analysis was performed using FBXO7 and anti-actin (as loading control) antibodies. (B) HEK-293T and SH-SY5Y cells were transfected with siRNA (FBXO7-specific or control), FBXO7 cDNA or not (−). After 24 hr, cells were treated with MPP+ (300 µM for HEK-293T or 2 mM for SH-SY5Y) and toxic effects of MPP+ were monitored at 24 hr by MTT assay.
Figure 7.
Flp-In SH-SY5Y cells with induced FBXO7 expression and neuronal phenotype.
(A) Western blot analysis of FBXO7-EGFP protein level using FBXO7 antibody and anti-actin antibody as loading control after 2 days induction with doxycycline (+ Dox) or not (- Dox). (B) Representative microscopic images of neuronal differentiated Tyr52 and Cys52 cells (for 21 days). Nuclei were counterstained with DAPI (blue). Neuronal total outgrowth was quantified in Tyr52 and Cys52 cells with induced differentiation for 7–21 days. Data are represented as the means ± S.D. of three separate experiments.