Journal: bioRxiv

Article Title: Proteomic profiling of primary cilia in the developing brain uncovers new regulators of cortical development

doi: 10.1101/2025.05.03.652041

Figure Lengend Snippet: ( A ) CKAP2-YFP is localized to the primary cilium in NIH3T3 cells. ( B ) Immunostaining with anti-CKAP2 antibody shows that endogenous CKAP2 is present in the cilia in multiple cell types, including NIH3T3 cells, SH-SY5Y and primary cultured radial glial cells. The cilium in the white dashed box is magnified and displayed at the right side. ( C ) En face views of whole-mount embryonic mouse brain following immunofluorescence staining with an anti-CKAP2 antibody. Endogenous CKAP2 is present in the primary cilia of E12.5 brains in both dorsal and ventral regions. ( D ) Quantification of CKAP2-positive cilia in ( C ). A total of 15 areas from 4 brains were quantified for the dorsal or ventral region. Data are presented as mean ± SD. Statistics: two-tailed Student’s t-test. **, p < 0.01. ( E ) Ckap2 shRNA significantly reduced Ckap2 gene expression. Ckap2 mRNA levels were measured by qPCR. Control (con) shRNA is a non-targeting scrambled shRNA. Data are presented as mean ± SD. Statistics: One-way ANOVA with multiple comparisons (Tukey test). **, p < 0.01; *, p < 0.05. ( F ) Quantification of ciliated cells in control and Ckap2 knockdown NIH3T3 cells. At least 100 cilia were quantified for each experimental condition. n=3 biological replicates. Data are presented as mean ± SD. Statistics: One-way ANOVA with multiple comparisons (Tukey test). ns, not significant. ( G ) Quantification of ciliary length in control and Ckap2 knockdown NIH3T3 cells. A least 100 cilia were quantified for each experimental condition. Experiment was performed 3 times with similar results. Data are presented as mean ± SD. Statistics: One-way ANOVA with multiple comparisons (Tukey test). ns, not significant. ( H ) Ckap2 knockdown attenuated SHH-induced Hh signaling. 3 days after lentivirus-mediated transfection of Ckap2 shRNA, cells were serum-starved for 24 h with or without SHH. Hh signaling activity was evaluated via qPCR measuring transcript levels of Gli1 . Data are shown as mean ± SD. Statistics: Two-way ANOVA with multiple comparisons (Tukey test). ****, p < 0.0001; **, p < 0.01.

Article Snippet: The TaqMan gene expression probes used were Hs05030235_g1 ( MARCKS ), Hs02786624_g1 ( GAPDH ), Mm01210070_g1 ( CKAP2 ), Mm00494645_m1 ( GLI1 ), Mm00436026_m1 ( PTCH1 ) and Mm99999915_g1 ( GAPDH ) to normalize the samples.

Techniques: Immunostaining, Cell Culture, Immunofluorescence, Staining, Two Tailed Test, shRNA, Gene Expression, Control, Knockdown, Transfection, Activity Assay

Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A FLAG-tagged version of the SneRING was overexpressed in U2OS cells using PEI MAX transfection. 24 h later, cells were infected with Sne at an MOI of 1. Non-transfected cells served as a control. 48 h p.i., samples were collected and immunoprecipitation was performed using FLAG-magnetic beads, followed by mass spectrometry analysis. The graphs show identified proteins, with significance (-log 10 p-value calculated by two-tailed T-test, n = 3) plotted against the log 2 fold change (log 2 FC) of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to non-transfected/infected controls (U2OS+Sne) (A) or of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to transfected/non-infected controls (U2OS+SneRING) (B) . Enriched proteins are labeled in red, reduced proteins are shown in blue, and grey represents unchanged proteins. Only host cell proteins are shown. (C) Samples were prepared, and immunoprecipitation was performed as in A. Input and elution fractions were analyzed by SDS-PAGE and western blot using antibodies against Sne heat shock protein SnGroEL (star indicates that the signal represents a cross-reactive band observed at 130 kDa), Mic60, CKAP4, Erlin2, PHB, Cox5a, Tom70, and FLAG. Mic60, mitochondrial contact site and cristae organizing system 60; CKAP4, cytoskeleton‐associated protein 4; Erlin2, ER Lipid Raft Associated 2; PHB, Prohibitin; Cox5a, cytochrome c oxidase subunit 5A; Tom70, translocase of the outer mitochondrial membrane 70.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Transfection, Infection, Control, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Two Tailed Test, Labeling, SDS Page, Western Blot, Membrane

Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A reaction containing purified recombinant SneRING, recombinant E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5b), ubiquitin, and ATP was mixed with the respective substrate (purified recombinant GST (A) or His-CKAP4 (B) ) for the indicated periods at 37 °C. Reactions without the ligase and/or substrate served as controls. Samples were analyzed by SDS-PAGE and western blot, using antibodies against SneRING, GST, ubiquitin, and CKAP4. Asterisks indicate ubiquitinated CKAP4. Ponceau staining of the western blot membranes is shown for the loading control.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Purification, Recombinant, Ubiquitin Proteomics, SDS Page, Western Blot, Staining, Control

Journal: Frontiers in Immunology

Article Title: A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer

doi: 10.3389/fimmu.2023.1264093

Figure Lengend Snippet: ΔNp63 regulates cancer cell uptake by macrophages via DKK3. (A) Analysis of the expression of the DKK3 gene in siΔNp63-transfected SCC90 cells. Data is represented as scatter plots with bars and mean +/- SEM (N=4). Two-tailed Student t-test: * p<0.05. (B) Analysis of the expression of the DKK3 protein in the cellular (upper panels) and supernatant (lower panels) fractions of siΔNp63-transfected SCC90 cells. (C) Analysis of the expression of the DKK3 protein in the cellular (left panels) and supernatant (right panels) fractions of SCC90 cells transfected with an anti-DKK3 siRNA. (D) Analysis of the in vitro phagocytosis of green-labeled SCC90 cells by red-labeled THP-1 macrophages upon siRNA-mediated DKK3. DAPI, Red cell dye, Green cell dye and merge are shown. White arrowheads indicate green-labeled phagocytosis vesicles. Magnification: X200. A magnification (right panels) of the inset in the merge is shown. (E) Quantification of the proportion (%) of THP-1 macrophages that display green-labeled phagocytosis vesicles. Data is represented as scatter plots with bars and mean +/- SEM (N=4). Two-tailed Student t-test: p=0.029. (F) Quantification of the proportion of THP-1 macrophages that display p65-positive nuclei. THP-1 cells were incubated with DMEM (negative control), 0.1 µg of TNF-α or 0.5 µg of hrDKK4 for 6 h prior to staining (see also Supplementary Figure S6A ). Data is represented as scatter plots with bars and mean +/- SEM (N≥3). ANOVA and Tukey post-test: *p<0.05; *** p<0.001. (G) Immunocytofluorescence analysis of the expression of p65 in THP-1 macrophages incubated with conditioned medium from siCtrl- (upper panels) or siDKK3- (lower panels) transfected SCC90 cells. DAPI, p65 staining and merge are shown. Magnification: X200. A magnification (right panels) of the inset in the merge is shown. White and yellow arrowheads highlight p65 staining in the cytoplasm and the nuclei, respectively. A quantification of the proportion of THP-1 macrophages with p65-positive nuclei is plotted in the graph. Data is represented as mean scatter plots with bars and +/- SEM (N=3). Two-tailed Student t-test: * p<0.05. (H) Western blot analysis of the expression of total and phosphorylated (pS473) AKT (left panels), and of total and phosphorylated (pS32) IκB-α in whole protein extracts from THP-1 cells incubated with rhDKK3 for 30 min and 2 h. AKT, AKT-pS473, IκB-α and IκB-α-pS32 signals were quantified with respect to the actin loading control and normalized to THP-1 macrophages incubated with DMEM (quantifications are shown). (I) Western blot analysis of CKAP4 expression in siCKAP4-transfected THP-1 macrophages. CKAP4 signals were quantified with respect to the actin loading control and normalized to siCtrl-transfected THP-1 macrophages (quantifications are shown). Shown blots are representative examples of three independent experiments. (J) A quantification of the proportion of siCtrl- and siCKAP4-transfected THP-1 macrophages with p65-positive nuclei upon incubation with 0.5 µg of rhDKK3 for 6 h. Data is represented as scatter plots with bars and mean +/- SEM (N=4). Two-tailed Student t-test: * p<0.05.

Article Snippet: CKAP4 downregulation was achieved by transfecting 15 nM of CKAP4 siRNA (Santa Cruz Biotechnology; sc-95758) to 5E 05 THP-1 cells using Lipofectamine RNAiMAX, according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Two Tailed Test, In Vitro, Labeling, Incubation, Negative Control, Staining, Western Blot, Control