Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr and immuno-stained with GT335, anti-acetylated tubulin and anti-Arl13b antibodies. ≥90 cells per sample were analyzed in three independent experiments. Error bars, S.D. ***p ≤ 0.001, **** p ≤ 0.0001. B ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immuno-stained with antibodies against GT335, Arl13b, Myo-Va, Cep89, Rab8a, Rabin8, IFT88, Cep290 and RPGRIP1L. Scale bar, 1µm. C ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A and B and visualized by staining with GT335 and antibodies against CP110. Scale bar, 1µm. N ≥ 90 cells per sample were analyzed in three independent experiments. Error bars, S.D. D ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immunostained with antibodies against Rab34 and Arl13b. Scale bar, 1µm. E ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A-C and immunostained with antibodies against GT335 and Arl13b. Scale bar, 1µm. F ) Primary cilia in sgRNA control and TBC1D19 knockout cells were examined by transmission electron microscopy (TEM) after 24 hr of serum starvation. Images of TBC1D19 knockout cilia from two consecutive sections are shown.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Staining, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) Control (sgCTL) and TBC1D19 knockout cells were treated with non-targeting control or CCP1 siRNA for 48 hr and serum starved for 24 hr, and extracts were subjected to immunoblotting with the indicated antibodies. B ) TBC1D19 knockout cells were infected with lentivirus expressing Flag-map4m or Flag-map4m-TTLL1 for 72 hr, serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. C ) Control (sgCTL), TBC1D19 knockout cells, and TBC1D19 knockout cells stably expressing TTLL5-EYFP or TTLL6-EYFP were serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. D ) Control (sgCTL), TBC1D19 -/- , C11ORF49 -/- , and TTLL1 -/- cells were serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. E ) sgCTL, C11ORF49 -/- , and LRRC49 -/- cells were serum starved for 24 hr, immuno-stained with antibodies against acetylated tubulin and INPP5E. N ≥ 50 cilia were counted per sample in two independent experiments. Error bars, S.D. ns, not significant. F ) 293T cells were co-transfected with GFP or GFP-TBC1D19 with mCherry-Arl13b for 32 hr and serum starved for 16 hr. Lysates were subjected to immunoprecipitation with GFP-trap beads and immuno-blotted with antibodies against GFP and mCherry.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Western Blot, Infection, Expressing, Stable Transfection, Staining, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr and immuno-stained with GT335, anti-acetylated tubulin and anti-Arl13b antibodies. ≥90 cells per sample were analyzed in three independent experiments. Error bars, S.D. ***p ≤ 0.001, **** p ≤ 0.0001. B ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immuno-stained with antibodies against GT335, Arl13b, Myo-Va, Cep89, Rab8a, Rabin8, IFT88, Cep290 and RPGRIP1L. Scale bar, 1µm. C ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A and B and visualized by staining with GT335 and antibodies against CP110. Scale bar, 1µm. N ≥ 90 cells per sample were analyzed in three independent experiments. Error bars, S.D. D ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immunostained with antibodies against Rab34 and Arl13b. Scale bar, 1µm. E ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A-C and immunostained with antibodies against GT335 and Arl13b. Scale bar, 1µm. F ) Primary cilia in sgRNA control and TBC1D19 knockout cells were examined by transmission electron microscopy (TEM) after 24 hr of serum starvation. Images of TBC1D19 knockout cilia from two consecutive sections are shown.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Staining, Transmission Assay, Electron Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL–CRT interaction up-regulates the association of CRT and LRP1 and downstream senescent-related signaling pathways in HSC. LX-2 cells were treated with CRT antibody or IgG 1 hour before the incubation of MBL and H 2 O 2 . ( A ) Representative photomicrographs of the SA–β-gal staining of LX-2 cells. ( B ) The distribution of cell cycle on the treated LX-2 cells with flow cytometry analysis. ( C ) The mRNA levels of senescence-related genes were assessed by quantitative reverse-transcription (qRT)-PCR analysis. ( D ) The association of CRT and LRP1 was determined by immunoblotting of immunoprecipitates in membrane fractions of LX-2 cells. ( E ) The association of LRP1, Rab8, and p110 was determined by immunoblotting of immunoprecipitates in cytoplasm fractions of LX-2 cells. ( F ) The p53, p21, mTOR, p-mTOR, AKT, p-AKT, and p110 protein levels were assessed by Western blot analysis. ( G ) The mRNA levels of fibrosis-associated genes were assessed by qRT-PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, Student t test or 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. The data shown represent 3 independent experiments. ATPase, adenosine triphosphatase; CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: PI3K antibody (21739-1-AP), Rab8 antibody (55296-1-AP), p53 antibody (60283-2-Ig), p21 antibody (10355-1-AP), p16 antibody (10883-1-AP), Bax antibody (50599-2-Ig), and Bcl2 antibody (12789-1-AP) were supplied by Proteintech (Chicago, IL).

Techniques: Protein-Protein interactions, Incubation, Staining, Flow Cytometry, Reverse Transcription, Quantitative RT-PCR, Western Blot, Membrane

Journal: Current biology : CB

Article Title: Synaptic-like Vesicles Facilitate Pioneer Axon Invasion.

doi: 10.1016/j.cub.2019.06.078

Figure Lengend Snippet: Figure 1. Synaptic-like Vesicles Accumulate in Pioneer DRG Growth Cones at the DREZ (A) Confocal z projections of DRG pioneer axon navigation in a Tg(sox10:syn-gfp); Tg(sox10:mcherry) animal from a 24-h time-lapse starting at 48 hpf. Syn-GFP vesicles accumulate in the growth cone upon DREZ contact. White arrows denote Syn-GFP concentrate. Dashed orange boxes denote DREZ. (B) Tracing of growth cone Syn-GFP intensity throughout navigation. Blue box denotes DREZ entry. Dashed line denotes SEM (n = 4 DRG). (C) Growth cone Syn-GFP intensities before, during, and after DREZ localization (n = 4 DRG). (D) Quantification of actin clusters in DREZ-located DRG pioneer axons containing vesicle puncta (n = 16 growth cones; n = 52 actin clusters). (E) Super-resolution DeSOS image of DRG pioneer axon at the DREZ in a Tg(sox10:lifeact-gfp) animal stained for Synapsin and Rab8. Dashed white box denotes single-plane insets on right. Arrowheads denote Lifeact-GFP, Synapsin, and Rab8 puncta in the pioneer growth cone. (F and G) Super-resolution DeSOS images of a DRG pioneer axon at the DREZ in a Tg(sox10:lifeact-gfp) animal stained for Synaptotagmin (F) and SV2 (G). Dashed white box denotes single-plane insets on right. Arrowheads denote colocalized Lifeact-GFP and vesicle puncta. Tukey’s HSD used for (C). SEM is shown. Scale bars, 10 mm. See also Figure S1 and Video S1.

Article Snippet: Primary antibodies used in this study are Synapsin 1/2 (1:1000, rabbit, Synaptic Systems, catalog #: 106 102 [59], Rab8 (1:500, mouse, Novus Biologicals, catalog #: H00004218-M02), RFP to detect tag-BFP (1:100, rabbit, Thermo Fisher, catalog #: R10367), Znp-1 (Synaptotagmin 2, 1:500, mouse, DHSB), SV2A (1:500, mouse, DHSB), MMP14 (1:500, rabbit, Sigma, catalog #: AB6005) [34].

Techniques: Staining

Journal: Current biology : CB

Article Title: Synaptic-like Vesicles Facilitate Pioneer Axon Invasion.

doi: 10.1016/j.cub.2019.06.078

Figure Lengend Snippet: Figure 5. MMP14a Localizes to Actin Clusters and Some Synaptic Vesicle Markers (A–C) Single-plane super-resolution, Imaris growth cone isolation images of DREZ-located growth cones in Tg(sox10:lifeact-gfp) animals stained for MMP14 and Synaptotagmin (A), SV2 (B), or Rab8 (C) at 56 hpf. Arrowheads denote MMP14 and/or vesicle puncta. (D) Quantification of actin clusters at the DREZ per growth cone that are MMP14+ and MMP14 (n = 41 growth cones). (E) Percent of actin clusters that colocalize with MMP14 and/or Synaptotagmin (n = 18 growth cones). (F) Confocal single-plane images of DREZ-located growth cones in Tg(sox10:syn-gfp) animals stained for MMP14. Arrowheads denote MMP14 puncta. (G) Percentage of MMP14 puncta that colocalize with vesicle markers. (H) Percentage of vesicle puncta that localize to MMP14 (n = 18 synaptotagmin stained; n = 8 SV2 stained; n = 15 Rab8-stained growth cones). (I–K) MMP14 intensity in Synaptotagmin- (I), SV2- (J), and Rab8 (K)-stained growth cones. Black bars denote actin-localized puncta. Gray bars denote puncta that do not localize to actin concentrates (n = 43 Synaptotagmin; n = 14 SV2; n = 5 Rab8 puncta). SEM is shown. Scale bars, 10 mm. See also Figure S3.

Article Snippet: Primary antibodies used in this study are Synapsin 1/2 (1:1000, rabbit, Synaptic Systems, catalog #: 106 102 [59], Rab8 (1:500, mouse, Novus Biologicals, catalog #: H00004218-M02), RFP to detect tag-BFP (1:100, rabbit, Thermo Fisher, catalog #: R10367), Znp-1 (Synaptotagmin 2, 1:500, mouse, DHSB), SV2A (1:500, mouse, DHSB), MMP14 (1:500, rabbit, Sigma, catalog #: AB6005) [34].

Techniques: Isolation, Staining

Journal: Scientific Reports

Article Title: Hepatocyte-secreted extracellular vesicles modify blood metabolome and endothelial function by an arginase-dependent mechanism

doi: 10.1038/srep42798

Figure Lengend Snippet: EVs secreted by primary rat hepatocytes were fractionated using a continuous sucrose density gradient. The fractions were assayed using Western blotting (A) with antibodies against indicated proteins and arginase activity (B) . Note: Arginase-1 protein and activity co-fractioned with exosomal markers Aip1/Alix, CD26, Rab8, CD81, CD63, and Hsp70.

Article Snippet: Mouse monoclonal antibodies were purchased from the following vendors: Mouse monoclonal antibodies against Aip1/Alix, Rab8 (clone 4/Rab8), and CD63 (clone AD1) were purchased from BD Biosciences; rabbit polyclonal antibody against CD26 was purchased from Abcam.

Techniques: Western Blot, Activity Assay