Journal: Breast Cancer Research : BCR

Article Title: Inhibition of cathepsin B activity attenuates extracellular matrix degradation and inflammatory breast cancer invasion

doi: 10.1186/bcr3058

Figure Lengend Snippet: Profile of cathepsin B, uPA, uPAR, β1-integrin and caveolin-1 expression in SUM149 and SUM190 cells . Expression of intracellular proteins was analyzed in duplicate samples of cell lysates, whereas expression of secreted proteins was analyzed in duplicate samples of conditioned media. Lysates and conditioned media of 2D cultures of SUM149 and SUM190 cells were equally loaded based on the protein concentration of the respective cell lysate and resolved by 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted with (a) an anti-cathepsin B (cat B) polyclonal antibody (bands represent proform (43 kDa), intermediate (38 kDa), single chain mature (31 kDa), and heavy chain (25/26 kDa) of double chain mature cathepsin B); (b) an anti-uPA antibody; (c) anti-uPAR, anti-β1-integrin, anti-caveolin-1 (cav-1) antibodies and (d) anti-β-tubulin monoclonal antibody with the concentration of α-tubulin in the cell lysate serving as a loading control for both cell lysates and their respective conditioned media. uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor.

Article Snippet: Cell lysates and conditioned media samples were equally loaded on the basis of the protein concentration of the respective cell lysates, separated by SDS-PAGE (10 or 12%) under either reducing or non-reducing conditions, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against human cathepsin B (1:4000; [ ]), β1-integrin (1:3000; a kind gift from Dr. Kenneth Yamada, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA), uPA (1:2000; Abcam, Cambridge, MA, USA), uPAR (1:2000; Abcam, Cambridge, MA, USA), caveolin-1 (1:4000; BD Biosciences, Bedford, MA, USA), or β-tubulin (1:1500; prepared from hybridoma E7 cells, Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, University of Iowa, Iowa City, IA, USA) and secondary antibodies conjugated with horseradish peroxidase (HRP; 1:10,000; Pierce) in TBS buffer (20 mM Tris, pH 7.5, 0.5 M NaCl) containing 0.5% Tween 20 and 5% (w/v) non-fat dry milk.

Techniques: Expressing, Protein Concentration, SDS Page, Membrane, Concentration Assay, Control

Journal: Cell

Article Title: Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis

doi: 10.1016/j.cell.2021.04.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-alpha tubulin , Cell Signaling Technology , Cat# 2125.

Techniques: Control, Virus, Recombinant, Western Blot, Giemsa Stain, Protease Inhibitor, Caspase-Glo Assay, Plasmid Preparation, Labeling, In Situ, Proximity Ligation Assay, Transfection, Magnetic Beads, Software, Cell Culture, Membrane

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: A Novel Ectodysplasin a Gene mutation of X-Linked Hypohidrotic Ectodermal Dysplasia

doi: 10.2147/CCID.S451125

Figure Lengend Snippet: (A) Real-Time Quantitative PCR:Compared with wild-type EDA, the mutant EDA protein significantly inhibited the expression of EDA level.****means p <0 .0001 (Student t test). ( B and C ) Protein expression of mutant EDA1 in transfected cell.HEK293T cells were transfected with vectors encoding mutant or wild-type soluble FLAG-tagged EDA1 protein, cell lysates were separately analyzed by western blotting, β-tubulin was used as a loading control. The bands showed that wild-type EDA1 can produce proteins, the weaker bands of p.M373I mutations in the cell lysates showed decreased intracellular protein expression. ****means p <0 .0001 (Student t test).

Article Snippet: The following antibodies were used: anti-EDA rabbit polyclonal antibody (Proteintech,1:1000 dilution), anti-β-tubulin (Cell Signaling,1:4000) and mouse anti-EDA (Cell Signaling,1:10,000 dilution).

Techniques: Real-time Polymerase Chain Reaction, Mutagenesis, Expressing, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Fibroblast-like TSPCs extracted from mice for subsequent experiments (scale bar 100 um). (b) Flow cytometry analysis of the expression of Pdgfra and Prrx1 (stem/progenitor cell surface markers) on TSPCs. (c) Immunofluorescence images of ARL13B (red) and α-tubulin (green) mark the cilia in shNC, shIFT88, and shARL3 TSPCs. DAPI staining indicates nuclei (blue), scale bar 10 um (upper) and 30 um (lower). (d) The percentage of ciliated TSPCs marked by ARL13B show the cilia counts in the control group and ciliary gene knockdown groups. (e) Cilia length in TSPCs with or without shIFT88 or shARL3 treatment (n= 25/group). (f) Representative images of alizarin red staining (upper) and alcian blue staining (lower) in TSPCs with or without shIFT88 or shARL3 treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (g) qPCR showed relative osteogenesis-related genes ( Alpl, Runx2, Bglap, Sp7 ) and chondrogenesis-related genes ( Col2a1, Sox9, Acan ) mRNA expression in TSPCs with or without shIFT88 or shARL3 treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. ns, P > 0.05.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining, Control, Knockdown, In Vitro

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) The volcano plot visualizes the differential expressed genes in injury site of the tHO mice 3 weeks after burn/tenotomy compared to the control mice. (b) RNA-seq performed between shIft88 and shNC TSPCs after 3-7 days of in vitro osteogenic differentiation and differential expressed genes are visualized. (c) Venn diagram shows the common differential expressed genes among three genesets and identified MEST, PTN, COL2A1 as the key genes. (d) The expression density of Mest in each cluster are shown by violin plot and UMAP plot. (e) qPCR shows relative Mest mRNA expression in TSPCs with or without shIFT88 or shARL3 treatment during in vitro osteogenic (left) or chondrogenic (right) differentiation. (f) Western blot shows MEST protein expression levels in TSPCs with or without shIFT88 or shARL3 treatment during in vitro osteogenic (left) or chondrogenic (right) differentiation. (g) UMAP plot shows the expression of Mest at different time points (Day0 ,3, 7, 21) during tHO development. (h) Immunohistochemistry images demonstrates MEST in tHO tendon tissues at different time post burn/tenotomy injury (scale bar 100 um). (i) Immunofluorescence images of α-tubulin (green) and MEST (red) in mouse primary TSPCs. DAPI staining indicates nuclei (blue), scale bar 10 um. Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Control, RNA Sequencing, In Vitro, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Representative images of alizarin red staining (upper) and alcian blue staining (lower) in TSPCs with or without siMest treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (b) qPCR shows relative Mest and osteogenic differentiation-related ( Alpl, Runx2, Bglap ) or chondrogenic differentiation-related genes ( Col2a1, Sox9, Acan ) mRNA expression in TSPCs with or without siMest treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (c) tSNE map shows Mest-high and Mest-low subclusters divided from Mesenchymal cluster. The expression level of Mest, Runx2, Sox9 , and Acan in Mest-high and Mest-low subclusters were visualize by violin plots. (d) Immunofluorescence images of α-tubulin (green) mark the cilia of TSPCs. DAPI staining indicates nuclei (blue), scale bar 10 um. Ciliated TSPCs proportion (cilia counts) marked by α-tubulin and cilia length (n= 25/group) in the siNC and siMest group. (e) Micro-CT images show tHO of lower limbs in shNC and shMest groups (n= 4/group). Quantification of tHO degree by bone volume (BV, mm^3) and bone mineral dendity (BMD, g/cc). Data are presented as means ± SD of three independent assays. Statistical analyses were performed by Student’s t-test for two-group comparison and one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. ns, P > 0.05.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Staining, In Vitro, Expressing, Immunofluorescence, Micro-CT, Comparison

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Immunofluorescence images of α-tubulin (green) marked cilia in ciliated and non-ciliated mouse primary TSPCs after cilia isolation. DAPI staining indicates nuclei (blue). (b) OPLS-DA plot shows the differences between NC (blue, n= 7) and SIM (green, n= 6) groups. (c) Circular diagram of the classification statistics of differential lipids. (d) Bubble chart of lipid classification. The horizontal axis represents the quantity range of lipids, and the vertical axis represents the logarithmic transformation of fold change. Each circle represents a lipid substance. The larger the circle, the higher the variable importance. Different colors represent the primary classification, and lipids of the same classification are arranged together. (e) The volcano plot visualizes the differential expressed lipids in SIM group compared to the NC group. (f) Relative abundance of sphingomyelin and triglyceride are significantly different in SIM and NC group. (g) KEGG enrichment analysis of differential expressed lipids between SIM and NC group (C: cellular processes; E: environmental information processing; H: human diseases; M: metabolism; O:organismal systems). (h) The expression density of Sgms2 in each cluster (left) and in Mesenchymal subcluster (right) are shown by UMAP plot. (i) UMAP plot shows the expression of Sgms2 in Mesenchymal subcluster at different time points (Day0 ,3, 7, 21) during tHO development.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Immunofluorescence, Isolation, Staining, Transformation Assay, Expressing

Journal: Cell Stem Cell

Article Title: Stage-Specific Transcription Factors Drive Astrogliogenesis by Remodeling Gene Regulatory Landscapes

doi: 10.1016/j.stem.2018.09.008

Figure Lengend Snippet:

Article Snippet: Immunoblotting was performed as described previously ( ) using following antibodies: mouse anti-GFAP (1:1000, G3893, Sigma), rabbit anti-CX43 (1:000, ab11370, Abcam), rat anti-ASCL1 (1:1000, MAB2567, R&D systems), rabbit anti-NES (1:1000, 839801, BioLegend), rabbit anti-OLIG2 (1:1000, AB9610, Milipore), rabbit anti-NFIA (1:1000, ab41851, Abcam), rabbit anti-RUNX2 (1:1000, sc-10758 X, Santa Cruz), rabbit anti-GAPDH (1:1000, ab9484, Abcam) and mouse anti-FLAG (1:1000, F1804, Sigma).

Techniques: Recombinant, Protease Inhibitor, Ligation, SYBR Green Assay, Plasmid Preparation, Software, Microscopy

Journal: eLife

Article Title: Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

doi: 10.7554/eLife.26215

Figure Lengend Snippet: ( A ) Representative image of a 3-day-old chick embryo neural tube stained with acetylated α-tubulin, phalloidin and N-Cadherin. ( A’–A’’’’ ) Magnification of the boxed region in ( A ). ( B ) En face imaging of neuroepithelial end-feet with acetylated α-tubulin and IFT88. ( B’–B’’ ) Magnification of boxed region in ( B ). ( C–C’ ) Another example as in ( B’ ). ( D–D’ ) End-foot stained with α-tubulin and γ-tubulin. ( E ) En face imaging of E12.5 mouse embryo spinal cord and cortex stained with acetylated α-tubulin and IFT88. ( F ) Stills of a neuroepithelial cell (dotted lines show cell outline) en face imaging from apical to more basal (left to right). Tissue explant stained for α-tubulin, N-Cadherin and phalloidin. ( G ) Neural progenitor cell expressing EMTB-GFP (and nuclear localised GFP from pCIG-Neurog2) imaged with SIM. The boxed region was magnified in ( G’–G’’’ ). Three different angles off the boxed region in G generated by 3D reconstruction. ( H ) Diagram of microtubule organization at the apical end-feet and relationship with the acto-myosin ring and the AJs. For all figures, images were captured by wide-field microscopy, unless otherwise stated. Scale bars, ( A ) ( B ) ( E ) ( G ) ( A’–A’’’’ ) 10 μm, ( B’–B’’ ) ( C–C’ ) ( D–D’ ) ( F ) ( G’–G’’’ ) 2 μm. 10.7554/eLife.26215.005 Figure 1—source data 1. Actin-tubulin co-alignment at the apical adhesion belt level.

Article Snippet: Primary antibody dilutions in blocking buffer (0.1% Triton-X-100% and 1% heat inactivated donkey serum, in PBS): Acetylated alpha tubulin (Sigma, T7451; RRID: AB_609894 ) 1:150, alpha tubulin (YL1/2) 1:200, alpha tubulin (Abcam, ab7291) 1:150, N-Cadherin (ThermoFisher, 13–2100; RRID: AB_2533007 ) 1:300, GFP (Abcam, ab6673; RRID: AB_305643 ) 1:500, IFT88 (Proteintech, 13967–1-AP; RRID: AB_2121979 ) 1:200, γ-tubulin (Sigma, T5326;RRID: AB_532292 ) 1:300, Drebrin (Abcam, ab11068; RRID: AB_2230303 ) 1:200.

Techniques: Staining, Imaging, Expressing, Generated, Microscopy

Journal: eLife

Article Title: Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

doi: 10.7554/eLife.26215

Figure Lengend Snippet: ( A ) Representative en face image of apical end-feet stained with α-tubulin, phalloidin and N-Cadherin. The yellow line is a representative region to measure fluorescence intensity at a single point on the adhesion belt. ( B ) Line graphs of normalised fluorescence intensity across the adhesion belt at a single position as in ( A ). Dashed lines indicate the AJs as defined by N-Cadherin. Scale bar, 5 μm.

Article Snippet: Primary antibody dilutions in blocking buffer (0.1% Triton-X-100% and 1% heat inactivated donkey serum, in PBS): Acetylated alpha tubulin (Sigma, T7451; RRID: AB_609894 ) 1:150, alpha tubulin (YL1/2) 1:200, alpha tubulin (Abcam, ab7291) 1:150, N-Cadherin (ThermoFisher, 13–2100; RRID: AB_2533007 ) 1:300, GFP (Abcam, ab6673; RRID: AB_305643 ) 1:500, IFT88 (Proteintech, 13967–1-AP; RRID: AB_2121979 ) 1:200, γ-tubulin (Sigma, T5326;RRID: AB_532292 ) 1:300, Drebrin (Abcam, ab11068; RRID: AB_2230303 ) 1:200.

Techniques: Staining, Fluorescence

Journal: eLife

Article Title: Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

doi: 10.7554/eLife.26215

Figure Lengend Snippet: ( A – A’’ , B – B’’ , F – F’’ , G – G’’ ) En face imaging of apical end-feet following treatment of chick embryo neural tube explants with Nocodazole (Noc) or Latruncuin-A (Lat-A). Boxed areas indicate how a line is drawn across the adhesion belt for measurement of fluorescence intensity. Letters next to the boxes refer to the corresponding line graphs. ( C, D, H, I ) Line graphs of normalised fluorescence intensity across the adhesion belt. For Nocodazole a distance of 4 μm and for Latrunculin-A 2 μm was measured. Boxed area represents the adhesion belt and the letter refers to the box plot quantifications from that area. Error bars = SEM. ( C’, D’, H’, I’ ) Box plots of the area under the curve (adhesion belt) from the line graphs. The median value, as well as the upper and lower quartiles are represented. T-test, ( C’ ) p<0.0001 (DMSO [Nocodazole control]: 210 measurements, 6 explants in 3 experiments; Nocodazole: 270 measurements, 8 explants in 3 experiments), ( D’ ) p=0.51 (DMSO [Nocodazole control]: 180 measurements, 6 explants in 3 experiments; Nocodazole: 244 measurements, 8 explants in 3 experiments), ( H’ ) p<0.0001 (DMSO [Latrunculin-A control]: 140 measurements, 5 explants in 2 experiments; Latrunculin-A: 213 measurements, 7 explants in 3 experiments) and ( I’ ) p<0.0001 (DMSO [Latrunculin-A control]: 140 measurements, 5 explants in 2 experiments; Latrunculin-A: 213 measurements, 7 explants in 3 experiments). When the entire curve is considered in ( D ), the area of the Nocodazole treatment is statistically larger than that of the DMSO treatment, p<0.0001. ( E, K ) End-foot area measurements for DMSO and small molecule treatments, as outlined by the N-Cadherin staining ( A’’’, B’’’, F’’’, G’’’ ). T-test, ( E ) p=0.73 (DMSO [Nocodazole control]: 276 measurements in 3 experiments; Nocodazole: 304 measurements in 3 experiments) and ( K ) p<0.0001 (DMSO [Latrunculin-A control]: 222 measurements in two experiments; Latrunculin-A: 334 measurements in 3 experiments). Error bars = SEM. ( J ) Normalised tubulin fluorescence following DMSO or Latrunculin-A treatment. T-test, p<0.0001 (DMSO: 110 measurements in 2 experiments; Latrunculin-A: 205 measurements in 3 experiments). Error bars = SEM, scale bars, 10 μm. 10.7554/eLife.26215.015 Figure 3—source data 1. Nocodazole vs DMSO control. 10.7554/eLife.26215.016 Figure 3—source data 2. Latrunculin-A vs DMSO control.

Article Snippet: Primary antibody dilutions in blocking buffer (0.1% Triton-X-100% and 1% heat inactivated donkey serum, in PBS): Acetylated alpha tubulin (Sigma, T7451; RRID: AB_609894 ) 1:150, alpha tubulin (YL1/2) 1:200, alpha tubulin (Abcam, ab7291) 1:150, N-Cadherin (ThermoFisher, 13–2100; RRID: AB_2533007 ) 1:300, GFP (Abcam, ab6673; RRID: AB_305643 ) 1:500, IFT88 (Proteintech, 13967–1-AP; RRID: AB_2121979 ) 1:200, γ-tubulin (Sigma, T5326;RRID: AB_532292 ) 1:300, Drebrin (Abcam, ab11068; RRID: AB_2230303 ) 1:200.

Techniques: Imaging, Fluorescence, Control, Staining

Journal: eLife

Article Title: Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

doi: 10.7554/eLife.26215

Figure Lengend Snippet: ( A ) Representative image of HH17-18 chick embryo neural tube labelled with antibodies to detect drebrin and acetylated α-tubulin and stained with phalloidin. Magnified boxed region shown in A'-A''' . End-foot of a neuroepithelial cell mis-expressing ( B ) Drebrin-mCherry and ( B' ) EMTB-GFP and stained with ( B'' ) phalloidin. ( C ) Representative line graphs of normalised fluorescence intensity at the level of the actin cable ( B–B'' ). ( D ) En face imaging of neuroepithelial end-feet electroporated with Drebrin-YFP and stained for phalloidin. Boxed areas are magnified. White arrowheads indicate Drebrin-YFP and phalloidin co-localisation. Scale bars, ( A ) 20 μm and boxed region 5 μm, ( B ) 2 μm, ( C ) 10 μm. 10.7554/eLife.26215.044 Figure 7—source data 1. Drebrin-mCherry / Actin / EMTB-GFP alignment.

Article Snippet: Primary antibody dilutions in blocking buffer (0.1% Triton-X-100% and 1% heat inactivated donkey serum, in PBS): Acetylated alpha tubulin (Sigma, T7451; RRID: AB_609894 ) 1:150, alpha tubulin (YL1/2) 1:200, alpha tubulin (Abcam, ab7291) 1:150, N-Cadherin (ThermoFisher, 13–2100; RRID: AB_2533007 ) 1:300, GFP (Abcam, ab6673; RRID: AB_305643 ) 1:500, IFT88 (Proteintech, 13967–1-AP; RRID: AB_2121979 ) 1:200, γ-tubulin (Sigma, T5326;RRID: AB_532292 ) 1:300, Drebrin (Abcam, ab11068; RRID: AB_2230303 ) 1:200.

Techniques: Staining, Expressing, Fluorescence, Imaging

Journal: eLife

Article Title: Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

doi: 10.7554/eLife.26215

Figure Lengend Snippet: The Z-stack section in which IFT88 appears largest was selected for measurement of acetylated tubulin ring (yellow dashed line around cell) and IFT88 labelled structure. Capped lines represent the diameter of the microtubule ring and IFT88. Scale bar: 1 μm.

Article Snippet: Primary antibody dilutions in blocking buffer (0.1% Triton-X-100% and 1% heat inactivated donkey serum, in PBS): Acetylated alpha tubulin (Sigma, T7451; RRID: AB_609894 ) 1:150, alpha tubulin (YL1/2) 1:200, alpha tubulin (Abcam, ab7291) 1:150, N-Cadherin (ThermoFisher, 13–2100; RRID: AB_2533007 ) 1:300, GFP (Abcam, ab6673; RRID: AB_305643 ) 1:500, IFT88 (Proteintech, 13967–1-AP; RRID: AB_2121979 ) 1:200, γ-tubulin (Sigma, T5326;RRID: AB_532292 ) 1:300, Drebrin (Abcam, ab11068; RRID: AB_2230303 ) 1:200.

Techniques:

Journal: Developmental cell

Article Title: The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3.

doi: 10.1016/j.devcel.2014.09.004

Figure Lengend Snippet: Figure 1. Identification of ARL6 as an Interactor of IFT27 (A–C) Murine IMCD3 cells stably expressing human IFT27, IFT27[K68A] (‘‘GTP-locked’’), or IFT27[T19N] (‘‘GDP-locked’’) tagged at the C terminus with a LAP tag (S-tag followed by a HRV3C cleavage site and GFP) were stained for IFT88 (red), acetylated tubulin (white), and DNA (blue). IFT27LAP variants were visualized through the intrinsic fluorescence of GFP. (A) Inset shows the individual fluorescence channels vertically offset from one another by three pixels. A yellow arrowhead points to the base of a cilium in the GFP channel of IFT27[T19N]LAP cells. Scale bar, 5 mm (main panels), 1 mm (insets). (B and C) Magnified views of cilia from IFT27LAP cells (B) or IFT27[T19N]LAP cells (C). Scale bar, 1 mm. In (C) endogenous mouse IFT27 was knocked down leaving human IFT27[T19N]LAP as the major IFT27 protein in those cells. See Figure S1C for control siRNA experiment. (D) Lysates were subjected to anti-GFP antibody capture and HRV3C (control, IFT27LAP) or TEV (LAPIFT88) cleavage elution before SDS-PAGE and silver staining (top) or immunoblotting (bottom). Asterisks indicate proteases used for cleavage elution. In parallel, the eluates were analyzed by mass spectrometry and the spectral counts for each IFT-B subunit are shown in the table on the right. Spectral counts from LAPIFT88 are the aggregate of three separate mass spectrometry experiments. Immunoblotting for IFT-B subunits (IFT88 and IFT57) and for ARL6 was conducted to confirm the mass spectrometry results. Immunoblotting for the S-tag that remains on IFT27LAP after HRV3C cleavage shows the amounts of all IFT27 variants recovered in the LAP eluates. See also Figure S1.

Article Snippet: Antibodies against the following proteins were used: actin (Sigma, #A2066), IFT88 (Proteintech), IFT57 (Proteintech), cMyc (9E10), GST (Life Technologies, #A-5800), GFP (Nachury et al., 2007), acetylated tubulin (6-11B-1), ARL6 (Jin et al., 2010), BBS5 (Proteintech), BBS4 (Nachury et al., 2007), and IFT27 (Keady et al., 2012).

Techniques: Stable Transfection, Expressing, Staining, Control, SDS Page, Silver Staining, Western Blot, Mass Spectrometry