human epithelial kidney hek293t cells  (ATCC)

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Characterization of purified proteins used in this study. (A) SDS-PAGE gel of GFP-CP110, CEP97-GFP, and CEP97^CP110-GFP, purified from HEK293T cells. Gels were stained with Coomassie brilliant blue R250. (B) Analysis of purified GFP-CP110, CEP97-GFP, and CEP97^CP110-GFP by mass spectrometry. (C) The proportion of fully blocked MTs with increasing concentrations of GFP-CP110 in in vitro reconstitution assays. n = 91, 28, 142, 105, and 140 MT plus ends for 5, 10, 20, 30, and 50 nM GFP-CP110. (D) A still image and a kymograph representing dynamic MT (blue) behavior in the presence of 50 nM CEP97-GFP (green, no binding). (E) Bar plot showing that CEP97-GFP does not affect the plus end blocking of dynamic MTs in vitro by GFP-CP110. The numbers of analyzed MTs are indicated on the bar plots. (F) SDS-PAGE of CPAP-N WT -mCh and CPAP-N MUT -mCh, purified from HEK293T cells. Gels were stained with Coomassie brilliant blue R250. (G) Analysis of purified CPAP-N WT -mCh and CPAP-N MUT -mCh by mass spectrometry. Source data are available for this figure: .

Article Snippet: Human embryonic kidney 239T (HEK293T) cells , ATCC , Cat #CRL-321.

Techniques: Purification, SDS Page, Staining, Mass Spectrometry, In Vitro, Binding Assay, Blocking Assay

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Characterization of the CPAP–CP110 interaction. (A and B) Schemes of CP110 and CPAP illustrating the deletion mutants used in this study. “+,” interaction between CPAP and CP110; “−,” no interaction between CPAP and CP110, and “+/−,” weak interaction between CPAP and CP110. For CP110, CC1, and CC2 are the coiled-coil domains. For CPAP, CC1, and CC2 are coiled-coil domains; PN2–3, the tubulin-binding domain ; MBD, the MT-binding domain; and G-box, glycine-rich C-terminal domain forming an antiparallel β-sheet . (C and D) Streptavidin pull-down assays with BioGFP-CP110 truncations as bait and full-length GFP-CPAP as prey. (E and F) Streptavidin pull-down assays with BioGFP-CPAP truncations as bait and full-length GFP-CP110 (E) or GFP-CP110 (581–991) (F) as prey. The assays in C–F were performed with extracts of HEK293T cells co-expressing the indicated constructs and BirA and analyzed by western blotting with anti-GFP antibodies. (G) SEC-MALS analysis of CPAP-CC1 (magenta line), CP110-CC2 (green line), and an equimolar mixture of CPAP-CC1 and CP110-CC2 (black line). (H) Scheme illustrating the mechanism for CPAP-CC1 and CP110-CC2 association. (I and J) CD spectra (I) recorded at 15°C and thermal-unfolding profiles (J) recorded by CD at 222 nm. Proteins and colors as in G. (K and L) SAXS analysis of the CPAP-CC1/CP110-CC2 heterodimer. (K) Solution X-ray scattering intensity over scattering angle from a 1:1 mixture (monomer equivalents) of CPAP-CC1 and CP110-CC2. The fit to the data yielding the interatomic distance distribution is shown with a black line. (L) Surface representation of the X-ray scattering volume of CPAP-CC1/CP110-CC2, at 32 ± 3 Å estimated precision, derived from averaging 22 particle models calculated by ab initio fit to the scattering data. Source data are available for this figure: .

Article Snippet: Human embryonic kidney 239T (HEK293T) cells , ATCC , Cat #CRL-321.

Techniques: Binding Assay, Expressing, Construct, Western Blot, Circular Dichroism, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Characterization of the mutations disrupting CP110–CPAP interaction. (A) Schematic representation of the domain organization of full-length human CPAP and CP110. The minimal regions CPAP and CP110 that interact with each other are indicated. The domain nomenclature is as in . (B and C) Chemical crosslinking followed by mass spectrometry of CPAP-CC1/CP110-CC2. (B) Schematic representations of parallel (left) and antiparallel (right) arrangements of CPAP-CC1 and CP110-CC2 chains in the CPAP-CC1/CP110-CC2 heterodimer. Predicted heptad repeats or H are indicated in each chain. Observed inter-protein crosslinks between residues of CPAP-CC1 and CP110-CC2 are indicated by thin lines. (C) Normalized inter-protein crosslinks observed between CPAP-CC1 and CP110-CC2 in the CPAP-CC1/CP110-CC2 heterodimer. The heptad a and d position residues are shown in bold and are underlined. The CPAP-CC1 and CP110-CC2 residues that were mutated in this study are highlighted with asterisks. (D) SEC-MALS analysis of CPAP-CC1 L149A/K150A (magenta dashed lines), CP110-CC2 (green solid lines), and an equimolar mixture of CPAP-CC1 L149A/K150A and CP110-CC2 (black solid lines). (E) Co-immunoprecipitation of CEP97^CP110-GFP as bait and CPAP-N-mCh WT or mutant as prey from HEK293T cells using anti-GFP antibodies. (F and G) Analytical SEC analysis of CPAP-CC1 and CP110-CC2 variants. (F) Analytical SEC analysis of CP110-CC2 (green solid line) and CP110-CC2 R656A/L659A (dark green–dashed line). (G) Analytical SEC analysis of CPAP-CC1 (magenta line), CP110-CC2 R656A/L659A (dark green–dashed line), and an equimolar mixture of CPAP-CC1/CP110-CC2 R656A/L659A (black solid line). Source data are available for this figure: .

Article Snippet: Human embryonic kidney 239T (HEK293T) cells , ATCC , Cat #CRL-321.

Techniques: Mass Spectrometry, Immunoprecipitation, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Key resources table

Article Snippet: Human embryonic kidney 239T (HEK293T) cells , ATCC , Cat #CRL-321.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Expressing, Software, Imaging

Journal: mBio

Article Title: Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination

doi: 10.1128/mbio.01221-24

Figure Lengend Snippet: Comparison of cellular activities of wild-type CNF1, CNF3, CNFy, and CNFx and chimeras of CNFx with CNF3 or CNFy. ( A ) Cellular dose-response curves of wild-type CNF1, CNF3, CNFy, and CNFx toxins and chimeric CNFx3(519), CNFxy(519), CNFyx(519), and CNFyx(688) toxins. HEK293T cells transfected with SRE-luciferase gene-reporter plasmids were treated with the indicated toxin at the indicated concentration for 6 h. Cells were then lysed and analyzed for cellular response by dual SRE-luciferase assay, as described in Materials and Methods. The data points shown are the mean values for that specified dose from four independent repeats performed in triplicate. Coomassie-stained SDS-PAGE analysis of purified recombinant proteins of wild-type CNF1, CNF3, CNFy, and CNFx toxins and chimeric toxins CNFx3, CNFxy, and CNFyx using the 519 joining site and CNFyx using the 688 joining site are shown in Fig. S2A. Corresponding scatter plots with all data points used to derive the best-fit lines and mean values are shown in Fig. S2B. ( B ) Schematic diagram depicting the wild type and chimeric toxin constructs generated to test swappability of domains among CNF3, CNFy, and CNFx, where the functional domains (delivery vehicle and cargo) and joining the site (position 519 or 688) of the CNF wild-type and chimeric proteins are indicated. Black = CNFx, red = CNFy, purple = CNF3, A = catalytic domain, B2 = location of the putative secondary binding domain in CNF1, DUF4765 = pfam motif, CNF-N = N-terminus of CNF with homology to PMT-N delivery vehicle.

Article Snippet: Human embryonic kidney epithelial HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco-Invitrogen, Grand Island, NY, USA), supplemented with 0.37% sodium bicarbonate, 100 U/mL penicillin-streptomycin (ThermoFisher Scientific), and 10% FetalPlex Animal Serum (AS; Gemini Bio-Products).

Techniques: Comparison, Transfection, Luciferase, Concentration Assay, Staining, SDS Page, Purification, Recombinant, Construct, Generated, Functional Assay, Binding Assay

Journal: mBio

Article Title: Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination

doi: 10.1128/mbio.01221-24

Figure Lengend Snippet: Comparison of RhoA protein modification by CNF1, CNFy, CNFx, and DNT. ( A ) Gel-shift assay of RhoA expressed alone or coexpressed with CNF1, DNT, CNFy, or CNFx toxin in E. coli BL21 cells. Bacterial co-expression of recombinant RhoA alone or with the indicated wild-type toxin was performed in triplicate, as described in Materials and Methods. Cell lysates were analyzed by Coomassie-stained SDS-PAGE gel (upper panel) and by western blot using anti-HA antibodies (lower panel). A mobility gel shift of the RhoA band upwards relative to the unmodified RhoA control (WT) indicates deamidation (DA), while a shift downwards indicates transglutamination (TG). ( B ) Gel-shift assay of cell lysates from HEK293T cells treated with 1.5 nM CNF1, 250 nM DNT, or 500 nM CNFx toxin. Transfected HEK293T cells expressing RhoA were treated without (control) or with the indicated toxin in DMEM, as described in Materials and Methods. Cell lysates were separated by SDS-PAGE and analyzed by western blot using first anti-RhoA antibodies (upper panel), then anti-β-tubulin antibodies as loading control (lower panel). ( C ) Representative dose response of RhoA modification by wild-type CNF1 (left panels) and CNFx (right panels) toxins in HEK293T cells using gel-shift assays. Shown are western blots using anti-RhoA antibodies (upper panel) or anti-β-tubulin antibodies (lower panel) of cell lysates from HEK293T cells treated with the indicated toxin concentrations. Additional repeats are shown in Fig. S3A and B, along with the scatter plot used for quantification to determine their respective EC 50 values for Rho modification (Fig. S3C).

Article Snippet: Human embryonic kidney epithelial HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco-Invitrogen, Grand Island, NY, USA), supplemented with 0.37% sodium bicarbonate, 100 U/mL penicillin-streptomycin (ThermoFisher Scientific), and 10% FetalPlex Animal Serum (AS; Gemini Bio-Products).

Techniques: Comparison, Modification, Gel Shift, Expressing, Recombinant, Staining, SDS Page, Western Blot, Control, Transfection

Journal: mBio

Article Title: Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination

doi: 10.1128/mbio.01221-24

Figure Lengend Snippet: Impact of CNFx Cys mutation (C1005S) on cellular activity, RhoA modification, and sensitivity to endosomal acidification. ( A ) Cellular dose-response curves for wild-type CNF1 and CNFx and the point mutants CNF1 (R832H), CNF1 (R832N), CNF1 (R832H, N862E), CNF1 (R832N, N862E), and CNFx (E857N) in HEK293T cells. Cells were treated with the indicated toxin concentration for 6 h, then lysed and analyzed by SRE-luciferase assay, as described in Materials and Methods. The data points shown are the mean values for that specified dose from three independent repeats performed in triplicate. Corresponding scatter plots with all data points used to derive the best-fit lines and mean values are shown in Fig. S4B. ( B ) Cellular dose-response curves for wild-type CNF1 and CNFx and the point mutants CNFx (C1005S) and CNFx (E857N, C1005S) in HEK293T cells. Cells were treated with the indicated toxin concentration for 6 h, then lysed and analyzed by SRE-luciferase assay, as described in Materials and Methods. Data points shown are the mean values for that specified dose from three independent repeats performed in triplicate. SDS-PAGE analysis of the recombinant wild type and point mutants purified from E. coli BL21 are shown in Fig. S8A. Corresponding scatter plots with all data points used to derive the best-fit lines and mean values are shown in Fig. S8B. ( C ) Representative dose response blots of RhoA modification by CNFx (C1005S) (right panels) and CNFx (E857N, C1005S) (left panels) toxins in HEK293T cells using RhoA gel-shift assays, performed as described in Materials and Methods. Shown are western blots using anti-RhoA antibodies (upper panel) or anti-β-tubulin antibodies (lower panel) of cell lysates from HEK293T cells treated with the indicated toxin concentrations. Additional repeats are shown in Fig. S8C and D, along with the scatter plot used for quantification to determine their respective EC 50 values for Rho modification (Fig. S8E). ( D ) Sensitivity of wild-type and mutant CNF toxins to endosomal acidification. Shown are dose–response curves to NH 4 Cl treatment of the wild-type CNF toxins (CNF3, CNFy, CNFx), chimeric toxins (CNFx3, CNFxy), and point mutants [CNFx (C1005S), CNFx (E857N, C1005S)] in the SRE-luciferase assay, as described in Materials and Methods. HEK293T cells were treated with NH 4 Cl for 30 min prior to treatment with the indicated toxins and assayed for SRE cellular response activity after 6 h. Relative activity indicates the fold activation compared with no-inhibitor treatment. Data points shown are the mean values for that specified dose from three independent repeats performed in triplicate. Corresponding scatter plots with all data points used to derive the best-fit lines and mean values are shown in Fig. S8F.

Article Snippet: Human embryonic kidney epithelial HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco-Invitrogen, Grand Island, NY, USA), supplemented with 0.37% sodium bicarbonate, 100 U/mL penicillin-streptomycin (ThermoFisher Scientific), and 10% FetalPlex Animal Serum (AS; Gemini Bio-Products).

Techniques: Mutagenesis, Activity Assay, Modification, Concentration Assay, Luciferase, SDS Page, Recombinant, Purification, Gel Shift, Western Blot, Activation Assay