Journal: Aging (Albany NY)

Article Title: Fbxo7 and Pink1 play a reciprocal role in regulating their protein levels

doi: 10.18632/aging.202236

Figure Lengend Snippet: Expression of Fbxo7 and its PD familial mutants led to an accumulation of Pink1. The Pink1-Flag plasmid alongside Fbxo7-Myc or its mutated plasmids were co-transfected to three different cell lines ( A : HEK293A, B : HeLa and C : SH-Sy5y). 24 hours after transfection, the cells were treated by 10 μM CCCP for 2 hours. The total cell lysates were subjected to Western blot analyses by anti-Flag antibody for Pink1-Flag, anti-Myc antibody for Fbxo7-Myc, and anti-β-Actin for β-Actin. After the relative level of FL-Pink1 and PF-Pink1 was obtained by normalizing of two forms of Pink1 to β-Actin respectively, the relative ratio of the two forms of Pink1 was obtained by normalization of the relative level of the two forms of Pink1 to the control only transfected with Pink1. The relative ratios were shown as mean ± SD; n = 3 independent experiments; individual 2-way ANOVAs with Tukey’s multiple comparisons test; ***p<0.001.

Article Snippet: Pink1 sgRNA sequences: 5’-CACCGGTGATGTCCCTGCATGGAGCTGG-3’and 5’-AAACCCAGCTCCATGCAGGGACATCACC-3’; Pink1 Hom1 primers: 5’-TCCCCGACCTGCAGCCCAGCTCATCTCCTGAGAGCAGATCTG-3’ and 5’-CCGGAACCTCCTCCGCTCCCCAGGGCTGCCCTCCATGAGCA-3’; Pink1 Hom2 primers: 5’-AGTTCTTCTGATTCGAACATCGAACATGGCATCCTCTGTGTC-3’ and 5’-TGGAGAGGACTTTCCAAGCCTTTACTGCATGTTGACGCT-3’; siRNAs for human Fbxo7 were purchased from Santa Cruz Biotechnology (sc-75010).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Fbxo7 and Pink1 play a reciprocal role in regulating their protein levels

doi: 10.18632/aging.202236

Figure Lengend Snippet: Expression of Fbxo7 and its PD familial mutants resulted in an accumulation of endogenous Pink1 in a Pink1-Flag KI cell line. ( A ) a schematic illustration of an engineering strategy for generation of a Pink1-Flag KI cell line. Pink1 Hom1: Pink1 homology arm 1; NeoR: neomycin resistant gene; Pink1 Hom2: Pink1 homology arm 2. ( B ) confirmation of expression of Pink1-Flag in the positive Pink1-Flag KI clones with an anti-Flag antibody. The cells were treated by CCCP and MG-132. ( C ) expression of Fbxo7 and its PD familial forms led to accumulation of PF-Pink1. The plasmids harboring Fbxo7 or the PD associated mutations in Fbxo7 were transfected into the Pink1-Flag KI cells. The total cell lysates were analyzed by Western blots with anti-Flag and anti-Myc antibodies. After the relative level of Pink1 protein was obtained by normalizing of Pink to β-Actin, the relative ratio of Pink1 was obtained by normalization of the relative level of Pink1 from Fbxo7 transfected samples to the control only transfected with Pink1. The relative ratios were shown as mean ± SD; n = 3 independent experiments; individual 2-way ANOVAs with Tukey’s multiple comparisons test; ***p = 0.0007. ( D ) KD of Fbxo7 caused a decrease of PF-Pink1. The Fbxo7 siRNA and control siRNA were transfected into the Pink1-Flag KI cells. 14 hours after transfection, the cells were treated by 10 μM MG-132 for 2 hours. The total cell lysates were analyzed by Western blots with anti-Flag and anti-Fbxo7 antibodies. After the relative level of Pink1 protein was obtained by normalizing to β-Actin, the relative ratio of Pink1 was obtained by normalization of the relative level of Pink1 from siFbxo7 transfected samples to the control transfected with siCtrl. The relative ratios were shown as mean ± SD; n = 3 independent experiments; unpaired 2-tailed Student’s t test; **p = 0.0058.

Article Snippet: Pink1 sgRNA sequences: 5’-CACCGGTGATGTCCCTGCATGGAGCTGG-3’and 5’-AAACCCAGCTCCATGCAGGGACATCACC-3’; Pink1 Hom1 primers: 5’-TCCCCGACCTGCAGCCCAGCTCATCTCCTGAGAGCAGATCTG-3’ and 5’-CCGGAACCTCCTCCGCTCCCCAGGGCTGCCCTCCATGAGCA-3’; Pink1 Hom2 primers: 5’-AGTTCTTCTGATTCGAACATCGAACATGGCATCCTCTGTGTC-3’ and 5’-TGGAGAGGACTTTCCAAGCCTTTACTGCATGTTGACGCT-3’; siRNAs for human Fbxo7 were purchased from Santa Cruz Biotechnology (sc-75010).

Techniques: Expressing, Clone Assay, Transfection, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Fbxo7 and Pink1 play a reciprocal role in regulating their protein levels

doi: 10.18632/aging.202236

Figure Lengend Snippet: Expression of Fbxo7 and its PD familial mutants had no effect on Pink1 ubiquitination. The Pink1-Flag plasmid along with Fbxo7-Myc or its mutated plasmids were co-transfected into HEK 293A cells. 24 hours after transfection, the cells were treated by 10 μM MG132 for 2 hours. The total cell lysates were subjected to immune-precipitation (IP) with anti-Flag antibody after 5-minute boiling. The IPed samples and the total cell lysates were analyzed by western blot with anti-ubiquitin antibody for ubiquitinated Pink1, anti-Flag antibody for Pink1-Flag, anti-Myc antibody for Fbxo7 and Bag2. The total cell lysates were analyzed by Western blots with anti-Flag and anti-Myc antibodies. After the relative level of ubiquitinated Pink1 protein was obtained by normalizing of ubiquitinated Pink to total Pink1, the relative ratio of ubiquitinated Pink1 was obtained by normalization of the relative level of ubiquitinated Pink1 from Fbxo7 or Bag2 transfected samples to the control transfected with Pink1 only (top panel from IP). The relative ratios were shown as mean ± SD; n = 3 independent experiments; individual 2-way ANOVAs with Tukey’s multiple comparisons test; ***p<0.001.

Article Snippet: Pink1 sgRNA sequences: 5’-CACCGGTGATGTCCCTGCATGGAGCTGG-3’and 5’-AAACCCAGCTCCATGCAGGGACATCACC-3’; Pink1 Hom1 primers: 5’-TCCCCGACCTGCAGCCCAGCTCATCTCCTGAGAGCAGATCTG-3’ and 5’-CCGGAACCTCCTCCGCTCCCCAGGGCTGCCCTCCATGAGCA-3’; Pink1 Hom2 primers: 5’-AGTTCTTCTGATTCGAACATCGAACATGGCATCCTCTGTGTC-3’ and 5’-TGGAGAGGACTTTCCAAGCCTTTACTGCATGTTGACGCT-3’; siRNAs for human Fbxo7 were purchased from Santa Cruz Biotechnology (sc-75010).

Techniques: Expressing, Ubiquitin Proteomics, Plasmid Preparation, Transfection, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Fbxo7 and Pink1 play a reciprocal role in regulating their protein levels

doi: 10.18632/aging.202236

Figure Lengend Snippet: Pink1 stabilized Fbxo7 in SNc of mouse brain. The different parts of brain tissues were dissected from WT and Pink1 KO female mice with age of 13-16 weeks. The homogenates were analyzed by western blot to check the protein level of Fbxo7 ( A – C : The protein level of Fbxo7 was normalized to β-Actin, then the relative level of Fbxo7 from WT was further normalized to that from KO). The relative ratios were shown as mean ± SD; n = 4 independent experiments; unpaired 2-tailed Student’s t test; **p = 0.0028 for SNc; p = 0.5588 for Stratium; p = 0.9763 for Cortex. ( D ) the mRNAs of Fbxo7 from SNc of WT and KO were analyzed by RT-qPCR. The relative ratios were shown as mean ± SD; n ≥ 5 independent experiments; unpaired 2-tailed Student’s t test; p = 0.392. ( E ) expression of Pink1 had no effect on Fbxo7 in HEK 293A cells. The relative ratios were shown as mean ± SD; n = 3 independent experiments; unpaired 2-tailed Student’s t test; p = 0.8255.

Article Snippet: Pink1 sgRNA sequences: 5’-CACCGGTGATGTCCCTGCATGGAGCTGG-3’and 5’-AAACCCAGCTCCATGCAGGGACATCACC-3’; Pink1 Hom1 primers: 5’-TCCCCGACCTGCAGCCCAGCTCATCTCCTGAGAGCAGATCTG-3’ and 5’-CCGGAACCTCCTCCGCTCCCCAGGGCTGCCCTCCATGAGCA-3’; Pink1 Hom2 primers: 5’-AGTTCTTCTGATTCGAACATCGAACATGGCATCCTCTGTGTC-3’ and 5’-TGGAGAGGACTTTCCAAGCCTTTACTGCATGTTGACGCT-3’; siRNAs for human Fbxo7 were purchased from Santa Cruz Biotechnology (sc-75010).

Techniques: Western Blot, Quantitative RT-PCR, Expressing