rabbit anti phf6 antibody  (Novus Biologicals)

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Overexpression analysis of variants in PHF6 from females vs males. ( a ) Protein scheme of PHF6 (Q8IWS0) with all missense variants previously published in male and female individuals , , , , , as well as three selected truncating variants , analysed via immunofluorescence coloured in red. Analysed variants (immunofluorescence) are printed in bold. Coding domains (according to uniprot ) are displayed in colour. NLS nuclear localization domain, NoLS nucleolar localization domain. ( b ) Table including all published missense variants occurring in male and female individuals, their VIPUR score, their localization in regard to ePHD and their origin (inherited/de novo). Variants p.(Met1Tyr) and p.(Ser138Cys) do not reside in a domain and cannot be modelled. Analysed variants (immunofluorescence) are printed in bold. ePHD extended atypical PHD-like zinc finger domain, 1 = N-terminal, 2 = C-terminal. ( c ) Immunofluorescence of HEK-293 cells transiently transfected with HA-tagged wildtype or mutant PHF6 co-stained with nucleolin (NCL). While wildtype PHF6 was expressed in the nucleoplasm as well as the nucleolus co-localizing with NCL, all three variants occurring in females led to an accumulation of PHF6 into aggregates, partly co-localizing with NCL. The truncating variant c.677delG (p.(Gly226Glufs*53)) resulted in small and more aggregates in the nucleus, and the two variants in the C-terminal ePHD resulted in large PHF6 aggregates while the expression of aberrant PHF6 in the nucleoplasm is reduced. In contrast, the three variants occurring in males resembled the wildtype with PHF6 expression in both the nucleoplasm and the nucleolus, co-localizing with NCL. Per variant, at least 40 cells were analyzed. Scale bar depicts 20 µm. Cells were stained with rabbit anti-HA (H6908, Sigma-Aldrich, 1:75) and mouse anti-nucleolin (39–6400, Thermo Fisher Scientific, 1:200). ( d ) Quantification of nuclear aggregates. The number of cells with or without aberrant large or small PHF6 aggregates in the nucleus was quantified for each variant and the wildtype. At least 40 cells were evaluated for each variant. ♀, variant identified in a female individual; ♂, variant identified in a male individual.

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: Over Expression, Immunofluorescence, Transfection, Mutagenesis, Staining, Variant Assay, Expressing

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Variants in PHF6 in female individuals lead to a loss of protein. In fibroblasts of female individuals with variants in PHF6 , only the wildtype PHF6 allele was expressed. ( a ) Immunofluorescence of fibroblasts of three individuals with either a duplication of exons 4 and 5, a missense variant c.914G>T (p.(Cys305Phe), or a truncating variant c.955C>T (p.(Arg319*) and one control individual. In the control, PHF6 expression could be observed in all fibroblast cells. In affected individuals, expression of PHF6 was present in only 50% of cells. Scale bar represents 20 µm. Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls. Affected individuals showed expression of PHF6 in 30–50% of cells, respectively. For each individual, at least 63 cells were evaluated. ( c ) Sanger sequencing of genomic DNA (gDNA) and of cDNA from RNA of affected individuals. In the individuals with a missense or a truncating variant respectively, both wildtype and mutant allele could be observed on gDNA level. On cDNA level, only the wildtype allele was present, thus indicating nonsense mediated mRNA decay. ( d ) Exemplary western blot from fibroblasts of control and affected individuals stained for PHF6 (Santa Cruz Biotechnology, sc-365237, 1:500) and Tubulin (ab7291, abcam, 1:10,000) as a housekeeping gene. PHF6 levels in patient fibroblasts were decreased. Grouped blots were cropped from different blots. Uncropped blots can be found in Supplementary Fig. . ( e ) Graphical analysis of three western blot replicates indicating 50%-70% of remaining PHF6 protein in patient fibroblasts. Error bars depict the standard deviation. Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: Immunofluorescence, Variant Assay, Expressing, Staining, Sequencing, Mutagenesis, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Transcriptome analysis of neuron-like PHF6 KO cells. ( a ) Heatmap displaying 1,338 deregulated genes between differentiated SK-N-BE (2) PHF6 KO and control cells with adjusted p value < 0.05 (FDR corrected). 626 genes were upregulated, and 712 were downregulated in PHF6 KO cells. Heatmap was created using the pheatmap package v.1.0.12 applying the standard settings (complete linkage method for hierarchical clustering for both columns and rows, scaling for rows). Blue = downregulated; red = upregulated. ( b , c ) Gene Ontology (GO) term analysis of ( b ) upregulated and ( c) downregulated genes depicting the top five GO terms for biological processes. ( d ) Comparison of deregulated genes after KO/KD of common chromatin remodelers and transcription factors with deregulated genes after PHF6 KO divided in up- and downregulated genes. Deregulated genes after KO/KD of either KDM5C ( Kdm5c KO mouse model, RNA sequencing of amygdala and frontal cortex) , the BAF complex (dKO Baf155/Baf170 mouse model, RNA sequencing of the pallium of E17.5 embryos) , BMI1 ( Bmi1 KO mouse model, RNA sequencing of whole brain) or TCF4 ( TCF4 KD SH-SY5Y neuroblastoma cells) expressed a highly significant overlap with up- and downregulated genes after PHF6 KO. Darkness of color correlates with significance. ( e ) Significant GO terms involved in neuron development and axonogenesis (p < 0.005). ( f ) Venn diagram depicting that deregulated genes were significantly enriched for genes implicated in neurodevelopmental disorders (NDDs). Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Overlap of deregulated genes in PHF6 KO SK-N-BE (2) cells with other deregulated gene sets.

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques:

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: PHF6 KO in cells reveals impaired neurite outgrowth, proliferation and migration. ( a ) Example of differentiating control and PHF6 KO cells transfected with mCD8-GFP. The cell with PHF6 KO had a longer primary neurite (axon) than the control cell. Scale bar depicts 20 µm. ( b ) Quantitative analysis of total neurite length, number of primary neurites and axon length (longest primary neurite) of day 10 differentiated PHF6 KO and control cells. PHF6 KO cells had significantly longer protrusions (total neurite length) and significantly more primary neurites. Additionally, axon length was also significantly increased in PHF6 KO cells. Of note, while control cells developed 1–2 neurites, some of the PHF6 KO cells had up to five neurites. For each cell line, at least 15 cells were evaluated per biological replicate. ( c ) After 8 days of differentiation, PHF6 KO cells showed significantly impaired proliferation in comparison to control cells. ( d ) Scratch assay showing that undifferentiated PHF6 KO cells migrated significantly slower than control cells. All experiments were performed in biological triplicates, except for the proliferation assay, which was performed in biological duplicates. Error bars depict the standard deviation. Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: Migration, Transfection, Wound Healing Assay, Proliferation Assay, Standard Deviation