Journal: Journal of Biological Chemistry

Article Title: Functional Interaction between the Scaffold Protein Kidins220/ARMS and Neuronal Voltage-Gated Na+ Channels

doi: 10.1074/jbc.m115.654699

Figure Lengend Snippet: FIGURE 3. Interaction between Kidins220 and Nav channels. A, endogenous Kidins220 and Nav channels associate in the adult mouse brain. Brain cortices were dissected from adult wild-type (C57BL6/J) mice, lysed, and immunoprecipitated by using anti-pan-Nav antibodies, or anti-GFP antibodies as control (ctrl). Samples were analyzed by SDS-PAGE and immunoblotting with anti-Kidins220 and anti-pan-Nav antibodies, as indicated. B, exogenously expressed Kidins220 and Nav channels associate in HEK293 cells. HEK293 cells were co-transfected with plasmids coding for HA-Kidins220 and Nav1.2, lysed, and immunoprecipi- tated with anti-Nav1.2 antibodies, or anti-GFP antibodies as control (ctrl). Samples were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-Nav1.2 antibodies, as indicated. tot, 50 g of total cortex or cell lysate. C, COS cells (C) and HEK293 cells (H) were transiently transfected with Nav1.2, lysed, and processed by SDS-PAGE. Nitrocellulose membranes were probed with anti-Nav1.2 and anti-pan-Nav antibodies, as indicated. Lysate from non-transfected cells (NT, 50 g) was run as a control. D, HEK293 cells were transiently transfected with Nav1.2, fixed after 48 h, and processed for immunocytochemistry with the indicated antibodies. Scale bar, 10 m. WB, Western blot.

Article Snippet: The following antibodies were used: polyclonal anti-Kidins220 (number ab34790, Abcam, Cambridge, UK), monoclonal anti-HA high affinity (11867423001, Roche Diagnostics GmbH), monoclonal anti-sodium channel (pan) (number S8809, Sigma Aldrich), polyclonal anti-Nav1.2 (number ASC-002, Alomone Labs Ltd., Jerusalem, Israel), polyclonal anti-GFP (number A11122, Invitrogen), and monoclonal antiGFP (number ab1218, Abcam).

Techniques: Immunoprecipitation, Control, SDS Page, Western Blot, Transfection, Immunocytochemistry

Journal: bioRxiv

Article Title: Lineage transcription factors co-regulate subtype-specific genes providing a roadmap for systematic identification of small cell lung cancer vulnerabilities

doi: 10.1101/2020.08.13.249029

Figure Lengend Snippet: (A) RNA-seq (log2 FPKM) showing expression of SCN3A and KCNB2 , in SCLC tumors (T) or cell lines (C) in the specified SCLC subtype or NSCLC. The line indicates the mean, and the asterisks indicate p-values for each sample compared to SCLC-A tumors. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (B) UCSC Genome Browser tracks showing ASCL1, NKX2-1, PROX1 and H3K27ac ChIP-seq signals in NCI-H2107 cells around the genes encoding SCN3A and KCNB2 . Black bars above the tracks indicate computationally called binding sites. (C-E) Immunoblots showing SCN3A and KCNB2 protein in NCI-H2107 cells, 72 hours post-transfection with control siRNAs or siRNA targeted against either ASCL1, NKX2-1 , or PROX1 (C), or SCN3A (D) or KCNB2 (E). (F) WST-1 assay for cell viability from cells from (D,E). Each data point represents a biological replicate, and error bars indicate SEM. ANOVA with Bonferroni’s multiple comparisons test was used to determine significant differences relative to control. ns; not significant. (G) Quantification of colony formation assays in soft agar using varying doses of KCNB2 inhibitor Quinine shows increased sensitivity of SCLC-A NCI-H889, compared to SCLC-N NCI-H524, and SCLC-P NCI-H526. (H) Histograms showing SCN3A+ cells are detected in live SCLC-A but not SCLC-N or SCLC-P cell cultures using an SCN3A extracellular domain specific antibody (red). The background fluorescence with the secondary antibody only is shown (gray). (I,J) RT-qPCR for SCN3A (I) and ASCL1 (J) mRNA from FACs isolated SCN3A+ (red) or SCN3A-(black) cells from mixtures of SCLC-A with SCLC-P or – N. Each data point represents a biological sample, error bars = SD around mean, unpaired t-test, * p<0.05, ** p<0.01.

Article Snippet: Cells were incubated with SCN3A antibody (Alomone Labs, Anti-SCN3A (Nav1.3) (extracellular), Cat #: ASC-023, 4.25 μg/ml) for 30 min on ice, washed 3 times with ice cold FACs buffer, incubated with Alexa Fluor 568-conjugated secondary antibody (Thermofisher, Catalog # A10042, 1:400) for 30 min on ice, washed 3 times as above, and resuspended in ice cold FACs buffer.

Techniques: RNA Sequencing, Expressing, ChIP-sequencing, Binding Assay, Western Blot, Transfection, Control, WST-1 Assay, Colony Assay, Sensitivity, Fluorescence, Quantitative RT-PCR, Isolation, Chromatin Immunoprecipitation, ChIP-chip, FACS, Knockdown

Journal: Nature Communications

Article Title: Ca 2+ -pumping by PMCA-neuroplastin complexes operates in the kiloHertz-range

doi: 10.1038/s41467-025-62735-5

Figure Lengend Snippet: a Activation curve of BK Ca channels recorded in whole-cell mode with 10 µM free Ca 2+ in the patch pipette (inset) is distinctly shifted to the right by co-expression of either PMC2-NPTN (red symbols, 12 cells) or NCX2 (green symbols, 14 cells). Activation curves determined in experiments with the indicated [Ca 2+ ] i in the pipette are given as calibration for nominal intracellular Ca 2+ concentrations (data from ). Lines are result of a Boltzmann function fitted to the data (mean ± SEM). Note the large shift of the activation curve indicating a reduction of [Ca 2+ ] i from pipette-delivered 10 µM (dark grey) to values around 0.1 µM (light grey) by both Ca 2+ -transporters. b Electron micrograph showing distribution of immunogold particles for NCX2 transiently expressed in CHO cells and bar graph (mean ± SEM of the 13 cells shown) denoting surface density of the NCX2 protein. Data for PMCA2 from Fig. were added for comparison. c Representative BK Ca -currents recorded with 0.1 mM EGTA as a cytoplasmic buffer in response to 1, 2, and 5 Ca 2+ influx pulses as in Fig. in CHO cells expressing Cav2.2 and NCX2. The current-response to 5 pulses obtained with PMCA2-NPTN from Fig. (trace in grey) was added for comparison. Inset: BK Ca -currents recorded upon a single 0.8 ms Ca 2+ influx pulse with NCX2 or PMCA2-NPTN. Note slightly faster decay of the BK Ca -currents in the presence of PMCA2-NPTN complexes. d Plot summarizing the time constants of the current decay determined in experiments as in ( c ); squares represent mean ± SEM of the indicated number of cells; data for PMCA2-NPTN were added for comparison.

Article Snippet: Replicas were digested at 60 °C in a solution containing 2.5 % SDS and 20% sucrose diluted in 15 mM Tris buffer (pH 8.3) for 48 h followed by 37 °C for 20 h. The replicas were washed in washing buffer comprising 0.05 % bovine serum albumin (BSA, Roth, Germany) and 0.1% Tween20 (Tw20, Roth, Germany) in 50 mM Tris-buffered saline (TBS) and then blocked in a solution containing 5% BSA and 0.1% Tween20 in TBS at room temperature (RT) for 1 h. Subsequently, replicas were incubated at 15 °C two overnights (O/Ns) in the following mixtures of primary antibodies in a solution containing 1% BSA and 0.1% Tween20 made up in TBS: (i) PMCApan (5F10, mouse /Ms/, 1:3000, Invitrogen, Cat. MA3-914) and Cav2.2 (rabbit, 1.67 μg/ml, Synaptic Systems, Cat. 152303) or (ii) NCX2 (SLC8A2, 0.5 μg/ml, Alomone, Cat. ANX-012).

Techniques: Activation Assay, Transferring, Expressing, Loading Density, Comparison, Patch Clamp

Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Identification of NCX isoforms. Identification of NCX1, NCX2 and NCX3 in porcine sperm. Representative immunoblots using sperm samples from different boars with A ) anti-NCX1, ( B ) anti-NCX2 and ( C ) anti-NCX3 antibodies, and their respective blocking peptides

Article Snippet: To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 μL of ProLongTM Glass Antifade Mountant with NucBlue (Hoechst 33342; ref. P36985, Invitrogen) in the dark.

Techniques: Western Blot, Blocking Assay

Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Immunolocalization of NCX1 isoform. Localization of NCX1 in the plasma membrane of porcine sperm ( A – C ), and after the peptide competition assay ( D – F ). NCX1 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Article Snippet: To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 μL of ProLongTM Glass Antifade Mountant with NucBlue (Hoechst 33342; ref. P36985, Invitrogen) in the dark.

Techniques: Clinical Proteomics, Membrane, Competitive Binding Assay, Staining, Immunofluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Western blotting analysis to measure SLC8A3, SLC24A2, SLC24A3 and SLC24A4 protein expression in RKO cells, treated as indicated. (B) CCK8 assays (C) Scratch wound healing (D) Colony formation assay of RKO cells. Treatment as indicated. * P < 0.05; ns stands for no significant change.

Article Snippet: This research used the following antibodies: anti-GAPDH (60004-1-lg, Proteintech, San Diego, CA, USA), anti-SLC8A3 (ANX-013, Alomone, Jerusalem, Israel), anti-SLC24A2 (ab192419, Abcam), anti-SLC24A3 (ab129776, Abcam, Cambridge, UK), anti- SLC24A4 (118992-1-AP, Proteintech, San Diego, CA, USA).

Techniques: Western Blot, Expressing, Colony Assay

Journal: bioRxiv

Article Title: Increased expression of developmental Nav1.3 promotes hippocampal CA3 hyperexcitability in early-stage 5xFAD mice

doi: 10.64898/2026.07.28.741298

Figure Lengend Snippet: a) Representative confocal images of dorsal hippocampus CA3 showing c-Fos (green, top), Nav1.3 + (red, middle), and merged channels (yellow, bottom) for WT and 5xFAD at 3 months (left) and 7 months (right). The inset on the top-left panel indicates the CA3 subregion delineated using the Allen Brain Atlas where c-Fos/Nav1.3 were quantified. Scale bar = 400 μm. b) Quantification of neuronal activation in CA3, shown as c-Fos + cells per 1000 μm 2 . c-Fos expression was higher in 3-month 5xFAD mice than in 3-month WT mice or 7-month 5xFAD mice (two-way ANOVA genotype x age interaction: F(1, 8) =12.0, p = 0.009; post-hoc Sidak: 5xFAD 3 months vs WT 3 months, p =0.015; 5xFAD 3 months vs 5xFAD 7 months, p = 0.049). c) Quantification of Nav1.3/c-Fos colocalization, shown as the percentage of c-Fos + neurons that are Nav1.3 +. The genotype x age interaction was not significant for colocalization, but there was a significant main effect of genotype, with higher Nav1.3/c-Fos co-expression at 3 months in 5xFAD mice vs age-matched WT controls (F(1, 8) = 11.8, p = 0.009); post-hoc Sidak’s comparisons indicate increased colocalization in 3-month 5xFAD mice relative to 3-month WT (p = 0.035). No other comparisons reached significance. Data are mean ± SD; n = 3 mice per group, 9 fields analyzed per mouse.

Article Snippet: An anti-Nav1.3 antibody (rabbit polyclonal; Alomone Labs #ASC-004; Jerusalem, Israel) was used, directed against an epitope in the C-terminal region of Nav1.3.

Techniques: Confocal, Activation Assay, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Increased expression of developmental Nav1.3 promotes hippocampal CA3 hyperexcitability in early-stage 5xFAD mice

doi: 10.64898/2026.07.28.741298

Figure Lengend Snippet: a) Schematic of mossy fiber terminal (red) contact (red arrowhead, right) on CA3 pyramidal cell dendritic spine (light blue). Inset (left) shows an enlarged representation of mossy terminal-CA3 spine contact b) Representative pre-embedding immuno-electron micrographs showing Nav1.3 in a mossy-fiber terminal (top, presynaptic) and a CA3 dendritic spine (bottom, postsynaptic). Purple arrowheads indicate silver-intensified immunogold (SIG) labeling for Nav1.3. Scale bar = 400 nm. c) Quantification of Nav1.3 SIG particles from 50 MFTs in WT and 5xFAD mice at 3 and 7 months. Data are mean ± SD; n = 3 mice per group. Two-way ANOVA revealed a significant genotype x age interaction (F(1, 8) = 7.68, p = 0.024). Post-hoc Sidak comparisons showed increased Nav1.3 SIG particles in MFTs from 3-month 5xFAD mice compared with 3-month WT mice (p = 0.007); no other pairwise comparisons reached significance. d) Quantification of postsynaptic Nav1.3 SIG particles per 50 CA3 spines per mouse per condition. Two-way ANOVA showed no significant effect of genotype p = 0.42, age p = 0.96, or interaction p = 0.58; n= 3 mice per group.

Article Snippet: An anti-Nav1.3 antibody (rabbit polyclonal; Alomone Labs #ASC-004; Jerusalem, Israel) was used, directed against an epitope in the C-terminal region of Nav1.3.

Techniques: Labeling, Transmission Electron Microscopy

Journal: bioRxiv

Article Title: Increased expression of developmental Nav1.3 promotes hippocampal CA3 hyperexcitability in early-stage 5xFAD mice

doi: 10.64898/2026.07.28.741298

Figure Lengend Snippet: (a-c) Representative confocal images showing GCaMP6s expression (green, a), Nav1.3 immunolabeling (red, b) and merged channel (yellow, c) in dorsal CA3 following viral delivery. Arrowheads indicate examples of GCaMP6s + neurons with reduced Nav1.3 labeling after Nav1.3-shRNA; panel C also shows the implanted fiber tract (dashed outline) used for photometry. Scale bar = 400 μm d) Quantification of CA3 calcium activity (AUC/1000 frames) in 3-month-old WT, 5xFAD, 5xFAD+scrambled-shRNA, and 5xFAD+Nav1.3-shRNA mice. 3-month WT and 5xFAD data from are replotted for comparison. Nav1.3 knockdown significantly reduced CA3 activity relative to both untreated 5xFAD and 5xFAD+scrambled controls. Data are mean ± SD; n = 6-7 mice per group. One-way ANOVA was used to assess treatment effects at 3 months (F (3, 21) = 5.63, p = 0.005), followed by post-hoc tests (Tukey) for pairwise comparisons: 5xFAD vs 5xFAD+Nav1.3-shRNA, p = 0.030; 5xFAD+scrambled vs 5xFAD+Nav1.3-shRNA, p = 0.032; WT vs 5xFAD, p = 0.047. WT vs 5xFAD+Nav1.3-shRNA, p = 0.987. WT and 5xFAD here are the 3-month subset re-analyzed by one-way ANOVA, so the WT and 5xFAD p-value differs from the full two-way model in ).

Article Snippet: An anti-Nav1.3 antibody (rabbit polyclonal; Alomone Labs #ASC-004; Jerusalem, Israel) was used, directed against an epitope in the C-terminal region of Nav1.3.

Techniques: Confocal, Expressing, Immunolabeling, Labeling, shRNA, Activity Assay, Comparison, Knockdown, Immunofluorescence

Journal: PLOS Biology

Article Title: The luminal ring protein C2CD3 acts as a radial in-to-out organizer of the distal centriole and appendages

doi: 10.1371/journal.pbio.3003519

Figure Lengend Snippet: (A) Confocal images of expanded U2OS centrioles immunolabeled for tubulin (magenta), NA14 (yellow), and one of the following proteins (green): SFI1, CP110, CEP162, CEP90, MNR, or OFD1. Bottom panels show corresponding top views. Scale bar: 100 nm. (B) Quantification of top-view diameters for distal centriolar components: tubulin ( n = 15, 171 ± 14 nm), NA14 ( n = 12, 88 ± 15 nm), and C-terminal C2CD3 ( n = 18, 76 ± 14 nm). Data from three independent experiments. (C) Axial positions of centriolar proteins relative to the distal tubulin signal: N-term C2CD3: n = 42, −24 ± 12 nm; Middle C2CD3: n = 37 −32 ± 10 nm; C-term C2CD3: n = 33, −30 ± 10 nm; NA14: n = 36, −30 ± 13 nm; SFI1: n = 32, −30 ± 13 nm; CEP162: n = 24, −4 ± 19 nm; CP110: n = 47, 28 ± 12 nm; CEP90: n = 36, −56 ± 17 nm; MNR: n = 56, −37 ± 11 nm; OFD1: n = 46, −48 ± 16 nm. Data from three independent experiments. (D) Confocal images of expanded U2OS centrioles co-stained for tubulin (magenta) and dual-labeled (green) for CEP90–NA14, C-terminal C2CD3–CEP90, NA14–MNR, or C-terminal C2CD3–MNR. Right panels highlight the relatively proximal localization of CEP90 (white arrows). Scale bar: 100 nm. (E) iU-ExM image of centrioles stained for tubulin (magenta), middle C2CD3, and CEP90 (green), used for angle measurement (white lines). Scale bar: 100 nm. (F) Measured angles from (E). From 111 angles and 3 independent experiments. Average: 158°3 ± 8.3. (G) Schematic depicting CEP90 localization on the A-microtubule, consistent with observed angles. (H) Integrative model of distal centriole architecture: C2CD3 (green), NA14 (mustard), DISCO complex (black/yellow/orange), SFI1-C (orange), and Cep135 (dark blue), highlighting the proximity between C2CD3 and the DISCO complex. (I–N) Microtubule displacement assay with percentage of GFP/mCherry positive cells with proteins localized to cytosol (Cyt.) or microtubules (MT) for each condition. U2OS cells were transfected with (I) MNR-mCherry (Cyt.: 0; MT: 100), (J) C2CD3-GFP (Cyt.: 100; MT: 0), (K) C2CD3-GFP and MNR-mCherry (Cyt.: 64.23 ± 11.02), (L) C2CD3-GFP, MNR-mCherry and mCherry-OFD1 (Cyt.: 49.83 ± 9.33; MT: 50.17 ± 9.33), (M) C2CD3-GFP, MNR-mCherry and CEP90 (Cyt.: 8.254 ± 7.211; MT: 91.75 ± 7.211), N) C2CD3-GFP, MNR-mCherry, mCherry-OFD1 and CEP90 (Cyt.: 20.40 ± 18.32; MT: 79.60 ± 18.32). Note that C2CD3 co-transfection with MNR alone or in combination with OFD1 is not sufficient to completely drive its relocalization on MTs. C2CD3 is relocalized on MNR decorated microtubules most efficiently in combination with CEP90 and/or CEP90 and OFD1. Scale bar: 10 µm. From three independent experiments performed for each condition. (O) Model illustrating how C2CD3 extends between the microtubule triplets to connect with the DISCO complex. The detailed statistics of all the graphs shown in the figure are included in the file.

Article Snippet: NA14 (SSNA1) , 11797-1-AP , Proteintech , NA , 1/250 , 1/250.

Techniques: Immunolabeling, Staining, Labeling, Transfection, Cotransfection

Journal: PLOS Biology

Article Title: The luminal ring protein C2CD3 acts as a radial in-to-out organizer of the distal centriole and appendages

doi: 10.1371/journal.pbio.3003519

Figure Lengend Snippet: (A) Expanded centrioles from control (siCTRL) and C2CD3-depleted (siC2CD3) cells stained for C2CD3 (green) and tubulin (magenta). Centriole length is significantly reduced in siC2CD3 ( n = 61, 200 ± 48 nm) compared to siCTRL ( n = 63, 437 ± 52 nm); **** P < 0.0001, unpaired t t est, 4 experiments. Centriole diameter was not significantly altered (siCTRL: n = 44, 115 ± 24 nm; siC2CD3: n = 44, 123 ± 27 nm; P = 0.0564). C2CD3 depletion efficiency: C2CD3-positive centrioles: siCTRL = 99.5% ± 1; siC2CD3 = 2.24% ± 2.66 ( n = 4 experiments, 30 centrioles/experiment). (B) Expanded centrioles from siCTRL and NA14-depleted (siNA14) cells stained for NA14 (green) and tubulin (magenta). Centriole length is modestly reduced (siNA14: n = 97, 373 ± 85 nm; siCTRL: n = 58, 413 ± 69 nm; *** P = 0.004). Diameter is also decreased (siNA14: n = 35, 104 ± 17 nm; siCTRL: n = 44, 115 ± 16 nm; ** P = 0.0041). NA14-positive centrioles: siCTRL = 100% ± 0; siNA14 = 15.38% ± 7.94 ( n = 4 experiments). (C–F) Quantification of NA14, SFI1, CEP135 distal dot, and Centrin distal dot signals at centrioles in siCTRL and siC2CD3 cells. Percentage of NA14-positive and NA14-negative centriole. For NA14-positive centrioles: siCTRL = 100% ± 0, siC2CD3 = 4.03% ± 4.98. N = 4 independent experiment (30 centrioles per experiment). Percentage of SFI1-positive and SFI1-negative centriole. For SFI1-positive centrioles: siCTRL = 97.91% ± 2.55, siC2CD3 = 11.48% ± 5.77. N = 3 independent experiment (30 centrioles per experiment). Percentage of CEP135 distal dot-positive and CEP135 distal dot-negative centriole. For CEP135 distal dot positive centrioles: siCTRL = 93.65% ± 2.78, siC2CD3 = 5.91% ± 1.43. N = 3 independent experiment (30 centrioles per experiment). Percentage of Centrin distal dot-positive and Centrin distal dot-negative centriole. For Centrin distal dot positive centrioles: siCTRL = 89.48% ± 3.35, siC2CD3 = 7.83% ± 2.88. N = 3 independent experiment (30 centrioles per experiment). (G–J) Quantification of C2CD3, SFI1, CEP135 distal dot, and Centrin distal dot signals in siCTRL and siNA14 cells. Percentage of C2CD3-positive and C2CD3-negative centriole. For C2CD3-positive centrioles: siCTRL = 100% ± 0, siNA14 = 97% ± 2.64. N = 3 independent experiment (30 centrioles per experiment). Percentage of SFI1-positive and SFI1-negative centriole. For SFI1-positive centrioles: siCTRL = 97.91% ± 2.50, siNA14 = 93.6% ± 6.25. N = 3 independent experiment (30 centrioles per experiment). Percentage of CEP135 distal dot-positive and CEP135 distal dot-negative centriole. For CEP135 distal dot positive centrioles: siCTRL = 91.31% ± 5.20, siNA14 = 95% ± 5.57. N = 3 independent experiment (30 centrioles per experiment). Percentage of Centrin distal dot-positive and Centrin distal dot-negative centriole. For Centrin distal dot positive centrioles: siCTRL = 89.48% ± 3.35, siNA14 = 92.15% ± 6.08. N = 3 independent experiment (30 centrioles per experiment). (K–N ) Quantification of SFI1, C2CD3, NA14, and CEP135 signals in siCTRL and siSFI1 cells. Percentage of SFI1-positive and SFI1-negative centriole. For SFI1-positive centrioles: siCTRL = 97.22% ± 2.54, siSFI1 = 37.33% ± 4,07. N = 3 independent experiment (30 centrioles per experiment). Percentage of C2CD3-positive and C2CD3-negative centriole. For C2CD3-positive centrioles: siCTRL = 100% ± 0, siSFI1 = 98.83% ± 2.02. N = 3 independent experiment (30 centrioles per experiment). Percentage of NA14-positive and NA14-negative centriole. For NA14-positive centrioles: siCTRL = 100% ± 0, siSFI1 = 99.67% ± 0.57. N = 3 independent experiment (30 centrioles per experiment). Percentage of CEP135 distal dot-positive and CEP135 distal dot-negative centriole. For CEP135 distal dot positive centrioles: siCTRL = 93.65% ± 2.78, siSFI1 = 52.42% ± 0.76. N = 3 independent experiment (30 centrioles per experiment). (O–Q) Quantification of CEP90, OFD1, and MNR signals at centrioles in siCTRL and siC2CD3 cells. Percentage of CEP90-positive and CEP90-negative centriole. For CEP90-positive centrioles: siCTRL = 98.47% ± 1.36, siC2CD3 = 11.8% ± 8.07. N = 3 independent experiment (30 centrioles per experiment). Percentage of OFD1-positive and OFD1-negative centriole. For OFD1-positive centrioles: siCTRL = 96.73% ± 1.20, siC2CD3 = 10.32% ± 4.94. N = 3 independent experiment (30 centrioles per experiment). Percentage of MNR-positive and MNR-negative centriole. For MNR-positive centrioles: siCTRL = 100% ± 0, siC2CD3 = 10% ± 2.19. N = 3 independent experiment (30 centrioles per experiment). (R) Schematic model summarizing the dependency of distal centriolar components on C2CD3. All centrioles are stained for tubulin (magenta) and the indicated protein (green). Scale bars: 200 nm. The detailed statistics of all the graphs shown in the figure are included in the file.

Article Snippet: NA14 (SSNA1) , 11797-1-AP , Proteintech , NA , 1/250 , 1/250.

Techniques: Control, Staining