Journal: Journal of Biological Chemistry

Article Title: Microglia-derived Pronerve Growth Factor Promotes Photoreceptor Cell Death via p75 Neurotrophin Receptor

doi: 10.1074/jbc.m402872200

Figure Lengend Snippet: FIG. 1. Microglial expression of neurotrophin mRNA and pro- tein. A, primary cultures of rat retina-derived activated microglial cells were tested for expression of NGF, BDNF, NT3, and NT4 using RT-PCR and DNA sequencing. Activated microglial cells generated PCR prod- ucts of 249, 198, 145, and 747 base pairs consistent with expected sizes of PCR products for rat NGF, NT4, NT3, and BDNF. B, CM were collected from microglial cultures in the presence of protease inhibitors composed of aprotinin 1 mg/ml, AEBSF 400 g/ml, leupeptin hemisul- fate 1 g/ml, and pepstatin A 1 g/ml, and processed for immunoblot- ting using chemiluminescence. Immunoblots of microglial CM showed solitary high molecular mass bands of 32-, 55-, 58-, and 36-kDa reactive bands for NGF, NT4, NT3, and BDNF, respectively.

Article Snippet: Rabbit polyclonal antibodies against NGF (sc-548), BDNF (sc-546), NT3 (sc-547), NT4 (sc-545), and -tubulin (sc-9104) were obtained from Santa Cruz Biotechnology and used at 1 g/ml while rabbit anti-pro-NGF IgG (30) was used at 1:1000.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Generated, Western Blot

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a Simplified cartoon of human DNA licensing. ATP binding and hydrolysis highlighted in orange. The addition of ORC6 in our reactions means that the ORC6-dependent pathway is expected to be dominant. Coloured bars identify complexes associated with each pre-RC assay wash type. b Schematic of pre-RC assay protocol. c Pre-RC assay with reactions assembled ± geminin (Gem FL ). Gem FL (*green) stabilises CDC6 (*red) and inhibits CDT1 ΔN (*blue) and ORC6 (*purple) interaction within LS elutions. With HS elutions, Gem FL inhibits MCM2-7 loading. d Quantification of MCM2-7 loading in the HS-reactions of ( c , lanes 3 and 4). Data normalised to the mean pre-RC reaction and analysed by two-tailed paired t-test (**p = 0.0042). Mean of three biological repeats ±SD. e In solution EM analysis of DH formation. 500–1000 micrographs were collected for each condition, and particles picked automatically. Two rounds of 2D classification were used to select for DHs (representative classes shown) and used to determine DH/micrograph. Mean of two independent reactions. f Anti-CDT1 western blot of LS pre-RC reactions ± Gem FL . g Quantification of CDT1 signal in ( f ). Data expressed as a percentage of the CDT1 input, normalised and compared to the control pre-RC mean and analysed by two-tailed paired t-test, **p = 0.0052. Mean ± SD of three biological repeats. h Exclusion of CDT1 ΔN from pre-RC reactions inhibits MCM2-7 loading (*red = CDC6, *blue = CDT1 ΔN and *purple = ORC6). Representative of three biological repeats. i Domain organisation of our standard CDT1 ΔN construct (residues 158–546), the CDT1 middle helical (CDT1 MHD , residues 158–396) and C-terminal helical (CDT1 CHD , residues 391–546) domains. j Pre-RC assay of the CDT1 MHD and CHD at 1× and 5× concentration (compared to CDT1 ΔN control). k Quantification of CDC6 signal in ( j ). Reactions compared to the −CDT1 ΔN control using one-way ANOVA with mixed-effects analysis and Dunnett’s multiple comparisons test. ****p < 0.0001 and *p = 0.0158. Mean ± SD. l Quantification of HS MCM2-7 loading in ( j ). Reactions compared to the -CDT1 ΔN control using one-way ANOVA with mixed-effects analysis and Dunnett’s multiple comparisons test. ****p < 0.0001 and *p = 0.0319. Mean ± SD of three biological repeats. Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Binding Assay, Two Tailed Test, Western Blot, Control, Construct, Concentration Assay

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a Substitution of CDT1 with a co-expressed CDT1-geminin complex (+CG, −CDT1 ΔN , lanes 3 and 6) inhibits pre-RC formation to a comparable extent as adding geminin (Gem FL , lanes 2 and 5). *red = CDC6, *purple = ORC6, *green = geminin, *blue = CDT1. CDT1 runs as a doublet due to proteolytic degradation. Representative of three biological repeats. b Titration of geminin into the pre-RC assay, showing that at least a 2× excess (1:2 CDT1 ΔN :Gem FL = CDT1 monomer to a geminin dimer) is required to inhibit DNA licensing. c Quantification of MCM2-7 loading from ( b ). No significant change in inhibition is observed when adding >2× geminin. Shown as the mean ± SD of three biological repeats, normalised to the pre-RC control and analysed using one-way ANOVA with Dunnett’s multiple comparisons test. **p = from bottom to top; 0.0011, 0.0017, 0.0032. d Mass photometry measurements of CDT1 ΔN (blue), geminin (Gem FL , green) and CDT1 + geminin (CDT1 ΔN + Gem FL , yellow) showing that CDT1 ΔN and geminin form a complex of ~90 kDa, that is consistent with the hetero-trimer (Tri., indicated by the grey shading). e Mass photometry exploring previously reported CDT1:geminin ratios (1:2 = hetero-trimer (yellow), 1:3 = hetero-tetramer (green), 1:4 = hetero-pentamer (blue), 2:4 = hetero-hexamer (red)). An excess of geminin results in reduced relative counts of the CDT1-geminin complex due to saturation of the movie frames with uncomplexed geminin dimers. The grey shading indicates the expected masses of each oligomeric state (abbreviated as Tri, Tet., Pent. and Hex.). f Expected molecular weights (M W ) of CDT1 ΔN , geminin and oligomeric complexes at the ratios detailed. Observed M W obtained from mass photometry measurements in parts ( d , e ) and from SEC-MALS of the CG complex from part ( a ). Geminin forms a dimer in solution (M W 48 kDa), and a hetero-trimer (1:2 CDT1 ΔN :geminin) is observed by mass photometry and SEC-MALS under all ratios tested. Data representative of two biological repeats and shown as the mean ± SE. Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Titration, Inhibition, Control

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a AlphaFold3 model of the CDT1-geminin hetero-trimer with CDT1 coloured blue and geminin green. Unstructured regions not shown. b–e The interaction regions identified by AlphaFold analysis. Geminin is shown in grey, with interaction regions shown in green, and CDT1 is shown in white with interaction regions in blue. Details of coloured residues are shown below the cartoons. Figures made using Chimera X. b Region 1: N-terminal CDT1 helix. c Region 2: CDT1 C-terminus with the N-terminal end of the geminin coiled-coil. d Region 3.1: the geminin coiled-coil. e Region 3.2: geminin N-terminal helix with the CDT1 middle helical domain. f Pre-RC assay comparing full-length CDT1 (CDT1 FL , residues 1–546), which contains the region 1 helix of interest shown in ( b ), and our standard CDT1 expression construct (CDT1 ΔN , residues 158–546) in which the helix of interest, along with the unstructured N-terminal extension, have been truncated to improve protein stability. g Quantification of MCM2-7 loading under high-salt (HS) conditions shown in part ( f ), showing no significant difference in MCM2-7 loading or inhibition of loading by geminin between the two CDT1 constructs. Data shown as the mean ± SD of three independent experiments, normalised to the mean of the CDT1 FL reaction and analysed by one-way ANOVA with Tukey’s multiple comparisons test, ****p < 0.0001. h Mass photometry of the middle helical domain (MHD) of CDT1 with geminin. Geminin results in a peak at ~48 kDa (green) and when CDT1 MHD is added to geminin, a secondary peak emerges at ~78 kDa, which is consistent with the predicted mass of a CDT1 MHD -geminin hetero-trimer (tri., expected M W 77 kDa, yellow). The MHD (M W 29 kDa) is below the limit of detection of ~30 kDa, therefore a gaussian fit has not been applied (blue). i Mass photometry of the C-terminal helical domain (CHD) of CDT1 with geminin. No secondary peak (predicted M W 67 kDa) is detected suggesting that the CDT1 CHD cannot bind to geminin. CDT1 CHD (M W 19 kDa) is below the limit of detection of ~30 kDa, therefore a Gaussian fit has not been applied (blue). Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Expressing, Construct, Inhibition

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a AlphaFold model of region 3.1—the geminin coiled-coil (residues of interest highlighted in green). Figure made using Chimera X. b Cartoon of coiled-coil peptides with the amino acid sequence above. Created in BioRender. Faull, S. (2025) https://BioRender.com/kxwwwdz . c Detailed kinetic and dissociation constants from SPR-determined binding data between immobilised CDT1 ΔN and coiled-coil mimetic peptides. *Indicates K D was determined via affinity modelling instead of kinetic fit. Number of replicates as per part ( d ), data shown as mean ± SE. d Summary of SPR-determined binding data between immobilised CDT1 ΔN and coiled-coil mimetic peptides. Values displayed as mean ± SE. N.B. = no binding. e Pre-RC assay of high-salt (HS) washed reactions comparing the inhibitory activity of geminin coiled-coil mimetic peptides to full-length geminin (Gem FL ). f Quantification of MCM2-7 bands from in part ( e ) compared to the control pre-RC reaction using one-way ANOVA with mixed-effects analysis and Dunnett’s multiple comparisons test. ****p < 0.0001, ns = not significant. Data shown as the mean ± SD. Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Sequencing, Binding Assay, Activity Assay, Control

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a AlphaFold model of interaction region 3.2. Geminin residues (76–95) shown in green and CDT1 interacting loop (318–333) in blue (Chimera X). b N-terminally elongated peptides. Created in BioRender. Faull, S. (2025) https://BioRender.com/whoj587 . c Kinetic and dissociation constants from SPR binding experiments. n = 3, except for Gem 90-145 n = 4. Mean ± SE. d Plot of SPR-determined K D values between immobilised CDT1 ΔN and geminin-mimetic peptides. Mean ± SE, n as per ( c ). e High-salt (HS) pre-RC assays of geminin and peptide derivatives. f Quantification of MCM2-7 band intensity from ( e ). Mean ± SD compared to control pre-RC using one-way ANOVA with mixed-effects analysis and Tukey’s multiple comparisons test (TMCT). ****p < 0.0001. ns = not significant. g Xenopus egg extract assay measuring DNA replication licensing by monitoring incorporation of [α− 32 P]-dATP into DNA relative to an untreated sample. To account for baseline variation, DNA synthesis of a mock-licensed negative control sample has been subtracted. Mean of two independent experiments. * = no DNA synthesis observed. h Pre-RC assay comparing the ability of chemically-synthesised peptide Gem 76–145 and recombinantly expressed recGem 76–145 to inhibit DNA licencing. Concentrations refer to the ratio of monomeric geminin to CDT1, 2x represents the CDT1-geminin hetero-trimer. n = 2 biological repeats. i Interaction between geminin (grey with residues 76–95 in green) and CDT1 (blue with residues 318–333 coloured by ΔΔG values from the BudeAlaScan web server). Side chains with the greatest ΔΔG are shown. j Mass photometry of CDT1 ΔN in which residues 327–330 have been mutated to alanines (CDT1 4A , green). Addition of CDT1 4A to geminin (Gem FL , control in blue) results in a complex peak consistent with a heterotrimer (yellow. Trimer = Tri., grey shading). Representative of n = 3. k Mass photometry of CDT1 R330A (green). Addition of CDT1 R330A to geminin (Gem FL , control in blue) results in a complex peak consistent with a heterotrimer (yellow). Representative of n = 3. l Pre-RC assay of CDT1 region 3.2 mutants. CDT1 4A reduces geminin-mediated pre-RC inhibition, but mutating residue 330 (CDT1 R330A ) alone is sufficient to disrupt the inhibition by geminin. m Quantification of HS MCM2-7 loading in ( l ). Mean ± SD of three biological repeats, compared to control pre-RC using one-way ANOVA with TMCT. ****p < 0.0001. Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Binding Assay, Control, DNA Synthesis, Negative Control, Inhibition, Residue

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a Alignment of the AlphaFold prediction of CDT1 (white) in complex with geminin (shown in cartoon form with tubular helices to highlight the C-terminus of the coiled-coil), with the human OCCM structure (PDB ID 8RWV). When aligning CDT1 in the AlphaFold structure with CDT1 in the human OCCM the steric clash between geminin and MCM2 (peach, shown in transparent surface view) is evident. Truncating the C-terminus of the geminin mimetic peptide reduces this clash. Figure made using Chimera X. b Cartoon of geminin peptides Gem 76-145 , Gem 76–140 , or Gem 76–135 . Created in BioRender. Faull, S. (2025) https://BioRender.com/3k0eyee . c Kinetic and dissociation constants for SPR experiments, n = 3, shown as mean ± SE. d Plot of K D values for binding of geminin peptides and immobilised CDT1 ΔN from SPR experiments. Data shown as the mean of 3 independent repeats ±SE. e Pre-RC HS-washed elutions after addition of Gem FL , Gem 76-145 , Gem 76–140 or Gem 76–135 to the pre-RC reaction. f Quantification of the mean MCM2-7 band intensity for reactions shown in ( e ) normalised to the control pre-RC reaction and compared mixed-effects analysis with Tukey’s multiple comparisons test. ****p < 0.0001. Data shown as the mean ± SD. Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Binding Assay, Control

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a Silver stained SDS-PAGE showing that the recruitment of CDT1 is inhibited by the addition of geminin to an ATPγS-containing pre-RC reaction. Representative of three biological repeats. b Corresponding Cy3 fluorescence emission of part ( a ) with fluorescently-labelled CDT1 (Cy3-CDT1 ΔN , annotated with *). c Two-step pre-RC reaction. The OCCM was assembled in the presence of ATPγS, after 18 min the control reaction (-Gem FL ) was washed and eluted. The other reactions were spiked with geminin for either 1, 2 or 4 min before washing. d Corresponding Cy3 fluorescence emission of part ( c ). e Quantification of Cy3-CDT1 fluorescence from part ( d ) showing CDT1 dissociation in the presence of geminin. Data shown as mean ± SD with a line of best fit of five biological repeats. f Alignment of CDT1-geminin AlphaFold model (shown in cartoon form) with the cryo-EM structure of the human OCCM (PDB ID 8RWV, shown in surface view) shows a steric clash between the C-terminus of the geminin coiled-coil (green) with MCM2 (peach). Figure made using Chimera X. g Proposed mechanism of how geminin inhibits DNA licensing by sequestering CDT1 away from the OCCM and preventing CDT1 from binding via steric hindrance. Source data are provided as a Source Data file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Staining, SDS Page, Fluorescence, Control, Cryo-EM Sample Prep, Binding Assay

Journal: Nature Communications

Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions

doi: 10.1038/s41467-025-67073-0

Figure Lengend Snippet: a Pre-RC assay using a combination of geminin and CDK/cyclin to assess effects on salt-stable MCM2-7 loading. b Quantification of high-salt stable loading from part ( a ). Data represented as the mean of three independent experiments ±SD. Relevant comparisons of conditions with and without geminin have been analysed using one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001, ***P = 0.0003. c Pre-RC assay testing the impact of pre-phosphorylation of pre-RC protein components with CDK1/cyclin A2 on salt-stable MCM2-7 loading. d Quantification of high-salt stable loading form part ( c ). Data represented as the mean of three independent experiments ±SD. Data were analysed using one-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001, ***p = from bottom to top; 0.0004, 0.0003, *p = 0.0167. e Pre-RC assay using a combination of pre-phosphorylation of pre-RC protein components with CDK1/cyclin A2 and the addition of geminin to assess the effects on salt-stable MCM2-7 loading. f Quantification of high-salt stable loading form part ( e ). Data represented as the mean of three independent experiments ±SD. Data were analysed using one-way ANOVA with Tukey’s multiple comparisons test, ****p < 0.0001, **p = 0.0024. g Graphical summary of pre-RC formation. In the presence of CDT1, DNA licencing can occur. The addition of geminin greatly inhibits DNA licensing activity, therefore co-operation between geminin and CDK is required for tight regulation. h Functional regions of geminin determined using mimetic peptides. The minimal CDT1 binding region (residues 96–135) are shown in pink. Residues 82–95 at the N-terminus of the peptide required to inhibit MCM2-7 loading through positioning of the coiled-coil are shown in teal and inhibitory residues at the C-terminus (136–145), needed for geminin dimerisation and to produce a steric clash with MCM2 in the OCCM, are shown in orange. Figure made using Chimera X. Source data are provided as a file.

Article Snippet: For the anti-CDT1 blots, pre-RC reactions were transferred onto PVDF membrane and blocked with milk in TBS-T prior to overnight incubation with an anti-CDT1 antibody (F-6, raised against residues 247-546, sc-365305, lot L2821, Santa Cruz, 1:1000).

Techniques: Phospho-proteomics, Activity Assay, Functional Assay, Binding Assay