fbxo11 antibody  (Bio-Techne corporation)

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Generation of a neuronal FBXO11 deficient cell model using CRISPR-CAS9 (A) Outline of generation process of FBXO11 heterozygous and complete knockout hIPSC lines is shown, including validation of mutated clones and isogenic controls. (B) Representative western blot of all nine FBXO11 hIPSC lines is shown. Blots were stained with anti-FBXO11 and anti-GAPDH antibodies. (C) Quantification of three independent western blot experiments of FBXO11 hIPSC lines confirmed loss of FBXO11 in HET and KO lines. Individual values are shown as dots and mean values are shown as bars with SEM. Mean of all three WT controls was set to 1. p values were calculated using a one-sample t test with a hypothetical control mean set to 1. For all HET and KO lines compared with the control mean, reduction was significant at p < 0.01. (D) Schematic outline of differentiation protocol from hIPSCs to NPCs and neurons with timeline and culture media used. Below, experiments performed here are indicated at various timepoints. (E) Representative images of immunofluorescence of FBXO11 WT, HET, and KO NPCs stained with antibodies against neural progenitor markers Nestin (NES, red) and PAX6 (green) confirm differentiation to NPCs. Images of all nine NPC lines can be found in Figure S2 . Images were taken on an AxioImager Z2 with Apotome 3 with a 40× objective. Scale bar, 20 μm. (F) Quantification of PAX6-positive cells among NPCs shows comparable levels of PAX6-positive cells for WT, KO, and HET cells. Quantification for individual lines can be found in Figure S3 . For quantification, cells were analyzed using CellProfiler identifying DAPI-positive cells and PAX6 positive cells (fraction of PAX6-positive cells = PAX6 stained cells/DAPI-stained cells).

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: CRISPR, Knock-Out, Biomarker Discovery, Clone Assay, Western Blot, Staining, Control, Immunofluorescence

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Gene expression changes due to loss of FBXO11 in human neurons and fly heads (A) Principal-component analysis (PCA) of three FBXO11 WT and three KO neuron samples showed clear separation of WT and KO samples along the first principal component. (B) Enriched gene ontology (GO) terms among differentially expressed genes were grouped based on function and show a broad enrichment of biological processes involved in, e.g., development, signaling, and migration. Detailed results on individual enriched GO terms can be found in Figure S5 . (C) Integration of GO term analysis of FBXO11-deficient human neuron and Drosophila head transcriptome analysis. The top five biological processes enriched in GO term analysis of human neurons are shown in black. The enrichment of these processes in Fbxo11-deficient Drosophila heads are shown in green. (D) Stacked bar chart grouping genes expressed in FBXO11 KO neurons based on their differential gene expression and colored by corresponding expression changes during neuronal differentiation in a publicly available dataset on gene expression during differentiation from hIPSC to neurons. Increasing expression during differentiation is marked in green, and decreasing expression during differentiation is shown in pink. Unchanged expression is shown in gray. Number of genes with increasing expression during differentiation is increased for genes downregulated in FBXO11 KO neurons. down = downregulated, up = upregulated, not sig = expression not significantly changed, exp. = expression.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Gene Expression, Migration, Expressing

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Loss of FBXO11 alters neuronal migration, proliferation, and differentiation (A) Representative images of neurosphere assay on FBXO11 WT, HET, and KO NPCs imaged 48 h after plating. Inner circle represents initial neurosphere size, outer circle represents migration after 48 h. Images were taken on a Nikon Ts2-FL microscope. Scale bar, 100 μm. (B) Quantification of migration as ratio between area occupied at 48 h and plating (0 h). Migration is impacted in HET and more severely in KO neurospheres. Quantification of all nine individual lines can be found in Figure S7 A. At least 30 neurospheres per genotype (≥8 neurospheres per line) from three independent experiments were analyzed. Significance was calculated using a Student’s t test. (C) Representative images of immunofluorescence of FBXO11 WT, HET, and KO NPCs stained with antibodies against proliferation markers Ki-67 (red) and mitotic marker pHH3 (green) are shown. Images were taken on an AxioImager Z2 with a 20× objective. Scale bar, 100 μm. (D) Quantification of Ki67-positive cells among NPCs shows increased levels of Ki67-positive HET and KO cells. Quantification for individual lines can be found in Figure S7 D. For quantification, cells from 15 images were analyzed using CellProfiler identifying DAPI-positive and Ki67 positive cells (fraction of Ki67-positive cells = Ki67 stained cells/DAPI-stained cells). Significance was calculated using a Student’s t test. (E) Proliferation of differentiating neurons (D20-D28) was assessed using an XTT assay. Absorbance was normalized to the absorbance of D20 (NPC stage and first day of measurement). Plotted is the mean (circle) together with a trend line (colored and dashed) and the standard error (gray shading). Proliferation differences between WT vs. HET and HET vs. KO were significant for all three tested timepoints (D23, D26, D28, p < 0.01) and between WT vs. HET for two timepoints (D23 and D26, p < 0.01). The experiment was carried out three times with three technical replicates each. Significance was calculated using a Student’s t test. (F) Representative western blot of 3-week-old neurons (D42) stained against neuronal marker MAP2, FBXO11, and H3 as a loading control. (G) Quantification of MAP2 levels from western blot in (F) showed reduced MAP2 levels (normalized to loading control H3) for HET and KO neurons compared with WT. The experiment was performed three times. Mean expression of three WT controls were set to 1. Significance was calculated using a one-sample t test with a theoretical mean of 1. (H) Representative images of immunofluorescence of FBXO11 WT, HET, and KO 1-week old neurons (D28) stained with antibodies against neuronal markers MAP2 (red) and TUBB3 (green) are shown here and images of all nine neuronal lines can be found in Figure S4 . Images were taken on an AxioImager Z2 with a 40× objective. Scale bar, 40 μm. (I) Quantification of TUBB3-positive cells among neurons showed reduced levels of TUBB3-positive KO cells. Quantification for individual lines can be found in Figure S7 F. For quantification cells from at least 15 images were analyzed using CellProfiler identifying DAPI-positive cells (all cells) and TUBB3-positive cells (fraction of TUBB3-positive cells = TUBB3-stained cells/DAPI-stained cells). Significance was calculated using a Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Migration, Neurosphere Assay, Microscopy, Immunofluorescence, Staining, Marker, XTT Assay, Western Blot, Control, Expressing

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Deficiency of Fbxo11 leads to impaired behavior and dendritic branching in Drosophila melanogaster (A) Climbing assay upon pan-neuronal knockdown of Fbxo11 showed impaired locomotor ability for two of the three RNAi lines tested. Individual data points are shown as circles and summarized data are shown as boxplots. At least 200 flies in batches of 10 ( n = 20) were analyzed per condition. Significance was calculated using a Wilcoxon signed rank test. (B) Representative image of traced da neurons from control and knockdown larvae upon da neuron-specific knockdown (477-Gal4; UAS-mCD8GFP driver line) is shown. Images were acquired using a Zeiss LSM 710 confocal microscope with a 20× objective. Scale bar, 100 μm. (C) Quantification of total dendrite length of da neurons. (D) Quantification of number of branches from da neurons. Tracings of da neurons were performed in ImageJ using the NeuronJ plugin. At least 10 da neurons from five different larvae from two independent crosses were analyzed for each line. Statistical significance was calculated using a Student’s t test.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Climbing Assay, Knockdown, Control, Microscopy

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Rescue of FBXO11-deficiency-associated phenotypes with proteasome-activating substances (A) Formulas of tested substances PD169316, R-Verapamil, and Verapamil are shown. (B) Scoring scheme for rescue experiments corresponding to the level of completeness of the rescue. For dark-filled boxes, rescue resulted in almost complete normalization to control levels under the DMSO (75%–100%). For boxes filled with light shades of respective color, rescue levels reached 50%–75%. Different tested substances were supplemented to the fly food or the cell culture medium and are color-coded as follows: black – DMSO control, green – PD169316, red – R-Verapamil, purple – Verapamil. For fly experiments, all substances were used at 1 μM, for cell-based experiments, different concentrations were used (PD169316: 20 μM, R-Verapamil: 15 μM, Verapamil: 10 μM). (C) Climbing assay deficit upon pan-neuronal Fbxo11 knockdown with RNAi 1 could partially be rescued with proteasome-activating substances supplemented to the fly food. Improvement of phenotypes was seen when adding substances at time of egg laying (developmental supp.) or after flies hatched (adult supp.). At least 200 flies in batches of 10 ( n = 20) were analyzed per condition. (D) Total dendrite length of da neurons increased upon addition of proteasome-activating substances to the fly food at time of egg laying. At least five different neurons from five different larvae were analyzed per condition. (E and F) NPCs (E) or D28 neurons (F) were treated with proteasome-activating substances for 2 days (NPCs) or 1 week (neurons) before staining with Ki-67 antibody to assess proliferation. For quantification, cells from at least 15 images were analyzed using CellProfiler identifying DAPI-positive and Ki67-positive cells (% of Ki67-positive cells = Ki67-stained cells/DAPI-stained cells). For all plots, individual data points are shown as circles and summarized data are shown as boxplots. Statistical significance was calculated using either a Wilcoxon signed rank test (climbing assay) or a Student’s t test (da neuron assays and cell-based experiments) with correction for multiple testing. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Control, Cell Culture, Climbing Assay, Knockdown, Staining