n ddk flag tag expression plasmid  (Sino Biological)

Journal: International Journal of Molecular Sciences

Article Title: Multi-Omics Studies Unveil Extraciliary Functions of BBS10 and Show Metabolic Aberrations Underlying Renal Disease in Bardet–Biedl Syndrome

doi: 10.3390/ijms23169420

Figure Lengend Snippet: Bbs10 deficiency is associated with an increase in cellular proliferation rate and enhanced basal ATP production. ( a ) IMCD- Bbs10 -/- showed an increase in proliferation compared to wild type cells. The mitotic index was calculated as the ratio of the number of cells undergoing mitosis to the total number of cells and expressed as a percentage. ( b ) Absolute quantification of cell survival was performed by measuring the absorbance at λ = 490 nm. The difference between the absorbance of wild type and IMCD3- Bbs10 -/- cells was highly significant, ( c ) The MTT assay demonstrates significantly increased cell viability in IMCD3- Bbs10 -/- cells compared to control cells. At a cell density of 0.5 × 10 4 cells/well, all three knockout cells showed significantly higher absorbance at λ = 570 nm after 24 h of growth. The assay was performed in triplicate and included three different IMCD3- Bbs10 -/- clones. ( d ) The ATP content in IMCD3- Bbs10 -/- cells and wild type cells at the indicated times after plating. ( e ) Measurement of ATP content in IMCD3- Bbs10 -/- cells and wild type cells treated in the presence of oligomycin for 5 h. Data are shown as the means ± SEM and are representative of three independent experiments, each performed in triplicate. In binary comparison, the significant difference was evaluated by performing a parametric t-test with a Welch correction in normally distributed datasets or Mann–Whitney test in not-normally distributed datasets. In multiple comparisons, the significant differences between groups were evaluated by performing an ordinary one-way ANOVA test and Hold–Sidak’s multiple comparison test in normally distributed datasets and Kruskal–Wallis test and Dunn’s multiple comparison test in not-normally distributed datasets. The normal distribution was verified according to the D’Agostino and Pearson test. (* p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant).

Article Snippet: After 24 h, cells were transfected with 10 µg of BBS10 cDNA ORF Clone, Human, N-DDK (Flag) tag expression plasmid (Sino Biological, SB) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the supplier instructions.

Techniques: MTT Assay, Knock-Out, Clone Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Multi-Omics Studies Unveil Extraciliary Functions of BBS10 and Show Metabolic Aberrations Underlying Renal Disease in Bardet–Biedl Syndrome

doi: 10.3390/ijms23169420

Figure Lengend Snippet: Bbs10 depletion affects energy metabolism in kidney-derived epithelial cells. ( a ) Relative mRNA expressions levels of key metabolic enzymes including Pdk1, Ldh1, Pkm2, Hk-1, Cpt-1, Cpt2, Acox-1, and Glut-1 (H) in IMCD3- Bbs10 -/- cells ( n = 20) and wild type cells ( n = 5). Data are presented as mean ± SEM and are normalized to wild type controls as the y -axis is indicated by the fold. The statistical significance of the difference in mRNA expressions between conditions (IMCD3- Bbs 10 -/- cells and wild type controls) was evaluated by an unpaired parametric t-test with Welch correction (* p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant). ( b ) Western blot analysis of the expression of hexokinase and Glut-1 in IMCD3- Bbs10 -/- cells and wild type. β-actin was used as the loading control. ( c ) Quantitative densitometric analysis was performed for data analysis and showed an increase in Glu-1 and Hk-1 also at protein levels ( p -value < 0.05). Data are shown as the means ± SEM and are representative of three independent experiments, each performed in triplicate.

Article Snippet: After 24 h, cells were transfected with 10 µg of BBS10 cDNA ORF Clone, Human, N-DDK (Flag) tag expression plasmid (Sino Biological, SB) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the supplier instructions.

Techniques: Derivative Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Multi-Omics Studies Unveil Extraciliary Functions of BBS10 and Show Metabolic Aberrations Underlying Renal Disease in Bardet–Biedl Syndrome

doi: 10.3390/ijms23169420

Figure Lengend Snippet: BBS10 protein-protein interaction (PPI) studies. ( a ) The BBS10 putative interactors were enriched for molecular processes in STRING (Search Tool for the Retrieval of Interacting Genes software). Clusters related to Cytoskeleton organization (GO:0007010, FDR 1.9 × 10 −3 ), Ribonuclease mrp complex (GO:0000172, FDR 2.2 × 10 −3 ), and BBSome-mediated cargo-targeting to cilium (HSA-5620922, FDR 3.1 × 10 −6 ) had results that were significantly enriched. ( b ) The more interesting BBS10-flag interactors were validated by Western blot analysis. The immunoprecipitates of the BBS10-flag were loaded on the SDS page. GFP-flag immunoprecipitates were used as a negative control. Each lane represents the immunoprecipitate derived from three different BBS10-flag cell clones. MID1: E3 ubiquitin-protein ligase Midline1; TAK1: also known as mitogen-activated protein 3 kinase 7 (MAP3K7); and TRIM65: Tripartite motif-containing protein.

Article Snippet: After 24 h, cells were transfected with 10 µg of BBS10 cDNA ORF Clone, Human, N-DDK (Flag) tag expression plasmid (Sino Biological, SB) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the supplier instructions.

Techniques: Software, Western Blot, SDS Page, Negative Control, Derivative Assay, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Multi-Omics Studies Unveil Extraciliary Functions of BBS10 and Show Metabolic Aberrations Underlying Renal Disease in Bardet–Biedl Syndrome

doi: 10.3390/ijms23169420

Figure Lengend Snippet: The primer sequences.

Article Snippet: After 24 h, cells were transfected with 10 µg of BBS10 cDNA ORF Clone, Human, N-DDK (Flag) tag expression plasmid (Sino Biological, SB) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the supplier instructions.

Techniques: