rabbit polyclonal foxp1 antibody  (Cell Signaling Technology Inc)

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) HEK293T cells were subjected to immunoprecipitation using ATR-specific antibody, the immunoprecipitates were analyzed via mass spectrum and FOXP1 was identified. ( B , C ) HEK293T cells were treated with 2 mM HU for 1 h or left untreated before immunoprecipitation using ATR ( B ) or ATRIP ( C ) specific antibodies. The immunoprecipitates were analyzed via immunoblotting using the indicated antibodies. ( D ) HEK293T cells transfected with a negative control siRNA or different siRNAs targeting FOXP1 were treated with HU for the indicated time before the whole cell lysates were harvested for immunoblotting with the indicated antibodies. ( E ) Upper panel: schematic of the DNA fiber assay examining stalled replication fork stability. Middle: representative images of CldU and IdU replication tracks. Lower panel: FOXP1 levels in different H1975 cells. ( F ) Statistical analysis of the IdU/CldU ratio of DNA fibers; the IdU/CldU ratio mean (red line) ±SD is shown. n , DNA fiber number, **** P < 0.0001; ns no significance; P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: siNC vs siFOXP1-1, 1.65e-024; siNC vs siFOXP1-2, 2.48e-018; siFOXP1-1 vs siFOXP1-2, 0.3593. ( G ) Control and FOXP1 knockdown H1975 cells were incubated with the indicated doses of HU for 6 h and then cultured for 14 d. The colonies were then stained with crystal violet and the percentage of viable cells were calculated. The mean percentage of viable cells (biological replicates, n = 3) ± SD is shown. * P < 0.05; ** P < 0.01; *** P < 0.001, P values were calculated by unpaired two-tailed t test. P value, shNC vs shFOXP1: 1 mM, 0.0098 ; 2 mM, 0.0011; 3 mM, 0.0320; 4 mM, 0.0011; 5 mM, 0.0008. ( H ) HEK293T cells transfected with a negative control siRNA or different siRNAs targeting FOXP1 were treated with 2 mM HU for the indicated time, and then subjected to chromatin fractionation. The protein levels in the whole cell lysate and chromatin fractions were examined via immunoblotting using the indicated antibodies. .

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Immunoprecipitation, Western Blot, Transfection, Negative Control, Control, Knockdown, Incubation, Cell Culture, Staining, Two Tailed Test, Fractionation

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) Coomassie blue staining of immunoprecipitates enriched with ATR-specific antibody. ( B ) His-tagged FOXP1 was incubated with GST-tagged ATRIP followed by GST pull-down assay. Proteins bound onto Glutathione beads were detected via immunoblotting using the indicated antibodies. ( C ) H1975 cells transfected with negative control siRNA or different siRNA targeting FOXP1 were treated with HU for 1 h before the whole cell lysates were harvested for immunoblotting with the indicated antibodies. ( D ) Statistical analysis of the IdU/CldU ratio mean in Fig. , mean (biological replicates, n = 3) ± SD is shown. *** P < 0.001; ns, no significance; P values were calculated by one-way ANOVA, followed by Dunnett’s test. P value: siNC vs siFOXP1-1, 0.0004; siNC vs siFOXP1-2, 0.0001; siFOXP1-1 vs siFOXP1-2, 0.9510. ( E ) HEK293T or H1975 cells transfected with siNC or siFOXP1 were pulse labeled with 10 μM BrdU for 30 min, followed by BrdU and PI staining and flow cytometric analysis. The percentage represents the BrdU-positive cells. ( F ) HEK293 cells transfected with negative control siRNA or different siRNA targeting FOXP1 were incubated with 100 nM CPT for 8 h or left untreated before harvested for PI staining and flow cytometric analysis. The percentage of S phase population was analyzed, mean (biological replicates, n = 3) ± SD is shown. **** P < 0.0001, P values were calculated by two-way ANOVA, followed by Sidak’s test. P value: siNC vs siFOXP1-1, 6.40e-006; siNC vs siFOXP1-2, 1.02e-006. ( G ) The whole cell lysates of HEK293T or H1975 cells transfected with siNC or siFOXP1 were harvested for immunoblotting with the indicated antibodies. ( H ) H1975 cells with different FOXP1 levels were subjected to sequential labeling with CldU and IdU for 30 min each, followed by DNA fiber assay. Left: representative images of CldU and IdU replication tracks. Middle: the IdU tract length was analyzed, IdU length mean (red line) ±SD is shown in scatter plot, n, DNA fiber number; ns, no significance; P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: siNC vs siFOXP1-1, P > 0.9999; siNC vs siFOXP1-2, P > 0.9999; siFOXP1-1 vs siFOXP1-2, P > 0.9999. Right: Mean IdU length ± SD (biological replicates, n = 3) was also analyzed and shown in column, ns, no significance, P values were calculated by one-way ANOVA, followed by Dunnett’s test. P value: siNC vs siFOXP1-1, 0.9898; siNC vs siFOXP1-2, 0.7224; siFOXP1-1 vs siFOXP1-2, 0.6490. ( I ) HEK293T cells transfected with FLAG-FOXP1 treated with 2 mM HU for 1 h were subjected to chromatin fractionation. Both the soluble and chromatin fractions were subjected to immunoprecipitation using a FLAG-specific antibody.

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Staining, Incubation, Pull Down Assay, Western Blot, Transfection, Negative Control, Labeling, Fractionation, Immunoprecipitation

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) Biotin-labeled ssDNA or complemented dsDNA were conjugated on streptavidin magnetic beads and incubated with GST-tagged FOXP1 purified from E. coli . Streptavidin-bound FOXP1 was detected via immunoblotting with the indicated antibodies. *, degraded GST-FOXP1. ( B ) Biotin-labeled ssDNA conjugated on streptavidin magnetic beads were incubated with GST-tagged FOXP1 or a GST-tagged FOXP1 Δ465–555 mutant purified from E. coli . Streptavidin-bound FOXP1 was detected via immunoblotting with the indicated antibodies. ( C ) Biotin-labeled ssDNA conjugated on streptavidin magnetic beads, with or without preincubation with His-tagged RPA70/RPA32, were incubated with GST-tagged FOXP1. Streptavidin-bound proteins were detected via immunoblotting with the indicated antibodies. ( D , E ) His-tagged RPA32 ( D ) or His-tagged-RPA70 (E) were incubated with GST-tagged FOXP1 before His pull-down assay. Proteins bound onto Ni beads were detected via immunoblotting with the indicated antibodies. ( F ) HEK293T cells treated with 2 mM HU for 1 h or untreated were subjected to immunoprecipitation using a RPA70-specific antibody. The immunoprecipitates were analyzed via immunoblotting with the indicated antibodies. ( G ) Proximity ligation assay experiments using FOXP1 and biotin-specific antibodies in H1975 cells. Lower: representative images of PLA foci. upper: quantification of the number of PLA foci per foci-positive cells (cell number: CON, n = 124; HU-1h, n = 95; HU-2h, n = 94), mean ± SD is shown. **** P < 0.0001, P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: CON vs HU 1 h, 7.20e-014; CON vs HU 2 h, 6.02e-027. ( H ) HEK293T cells were labeled with EdU followed by analysis by iPOND assay and immunoblotting with the indicated antibodies. .

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Labeling, Magnetic Beads, Incubation, Purification, Western Blot, Mutagenesis, Pull Down Assay, Immunoprecipitation, Proximity Ligation Assay

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) Biotin-labeled random dsDNA or dsDNA containing GTAAACA consensus motif was conjugated on streptavidin magnetic beads and incubated with GST-tagged FOXP1 purified from E. coli . Streptavidin-bound FOXP1 was detected via immunoblotting with the indicated antibodies. ( B ) Biotin-labeled random ssDNA or ssDNA containing GTAAACA was used to test FOXP1 binding as described in ( A ). ( C ) Schematic of full-length FOXP1 and its deletion mutants. ( D ) HEK293T cells transfected with FLAG-FOXP1 or its deletion mutants were subjected to immunoprecipitation using a FLAG-specific antibody. FOXP1 bound RPA proteins were detected via immunoblotting. ( E , F ) His-tagged RPA32 ( E ) or RPA70 ( F ) were incubated with GST-tagged FOXP1 (or its deletion mutants) followed by GST pull-down assay. Proteins bound onto Glutathione beads were detected via immunoblotting using the indicated antibodies. ( G ) Proximity ligation assay experiments using FOXP1 and biotin-specific antibodies in H1975 cells transfected with a negative control siRNA or siRNAs targeting RPA32 or RPA70. Upper panel: quantification of the number of PLA foci per foci-positive cells (cell number: siNC CON, n = 103; siRPA70-CON, n = 102; siRPA32-CON, n = 102; siNC HU, n = 110; siRPA70-CON, n = 106; siRPA32-CON, n = 106), mean ± SD is shown. ** P < 0.01, **** P < 0.0001, P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: siNC CON vs siRPA70-CON, 1.39e-018; siNC CON vs siRPA32-CON, 0.0039; siNC HU vs siRPA70 HU, 5.80e-038; siNC HU vs siRPA32 HU, 1.41e-010. Lower panel: representative images of PLA foci and immunoblotting of RPA expression.

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Labeling, Magnetic Beads, Incubation, Purification, Western Blot, Binding Assay, Transfection, Immunoprecipitation, Pull Down Assay, Proximity Ligation Assay, Negative Control, Expressing

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A , B ) HEK293T cells transfected with FLAG-tagged FOXP1 ( A ) or SFB-tagged OGT ( B ) were subjected to immunoprecipitation using a FLAG-specific antibody. Proteins in the immunoprecipitates were detected via immunoblotting. * a shorter isoform of FOXP1. ( C ) O-GlcNAcylation stoichiometry of FLAG-tagged FOXP1 was analyzed and examined via immunoblotting with FLAG-specific antibody. ( D ) HEK293T cells transfected with GFP-tagged OGT and full-length FLAG-FOXP1 or its deletion mutants were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. ( E ) O-GlcNAcylation stoichiometry of FLAG-tagged FOXP1 was analyzed and examined via immunoblotting with FLAG-specific antibody, with or without incubation with 2 mM HU for 1 h before cells were harvested. ( F ) H1975 cells transfected with FLAG-tagged FOXP1 preincubated with Thiamet-G or not were subjected to immunoprecipitation and immunoblotting using the indicated antibodies.

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Transfection, Immunoprecipitation, Western Blot, Incubation

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) HEK293T cells transfected with FLAG-tagged FOXP1 and increasing concentrations of GFP-tagged OGT, were subjected to immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were examined via immunoblotting using the indicated antibodies. ( B ) HEK293T cells preincubated with Thiamet-G (5 μM, 24 h) or not were subjected to immunoprecipitation using a FOXP1-specific antibody. The immunoprecipitates were examined via immunoblotting using the indicated antibodies. ( C ) GST-tagged FOXP1 was purified from bacteria expressing GST-FOXP1 along with His-OGT or the corresponding empty vector. GST-FOXP1 and its O-GlcNAcylation were detected via immunoblotting with the indicated antibodies. ( D ) HEK293T cells transfected with GFP-tagged OGT and wild-type FLAG-tagged FOXP1 or its deletion mutants were subjected to immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were examined via immunoblotting using the indicated antibodies. ( E ) His-tagged OGT was incubated with wild-type GST-tagged FOXP1 or its mutant lacking AA 556–610, before performing a GST pull-down assay. Proteins bound onto glutathione beads were detected via immunoblotting using the indicated antibodies. ( F ) HEK293T cells treated with 2 mM HU for the indicated time were subjected to immunoprecipitation using a FOXP1-specific antibody. The immunoprecipitates were examined via immunoblotting using the indicated antibodies. ( G ) HEK293T cells transfected with FLAG-tagged FOXP1 and GFP-tagged OGT were treated with 2 mM HU for the indicated time and subjected to immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were examined via immunoblotting using the indicated antibodies. ( H ) Negative control or OGT knockdown HEK293T cells were transfected with FLAG-tagged FOXP1 and treated with 2 mM HU for the indicated time before immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were examined via immunoblotting using the indicated antibodies. ( I ) GST, GST-tagged FOXP1, or GST-tagged FOXP1 modified with O-GlcNAc were incubated with HEK293T cell lysate before GST pulldown. The glutathione bead-bound signals were detected by immunoblotting. * non-specific signal. .

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Transfection, Immunoprecipitation, Western Blot, Purification, Bacteria, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Pull Down Assay, Negative Control, Knockdown, Modification

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A , B ) HEK293T cells transfected with FLAG-FOXP1 and HA-ATRIP treated with 2 mM HU for 1 h in the presence of ATR inhibitors VE-822 (1 μM) and NU6027 (10 μM) ( A ) or CHK1 inhibitors Rabusertib (5 μM) and UCN-01 (50 nM) ( B ), were subjected to immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were analyzed via immunoblotting using the indicated antibodies. ( C ) HEK293T cells overexpressing FLAG-tagged FOXP1 were treated with 2 mM HU for 1 h in the presence of the CHK1 inhibitor Rabusertib or not, before the cells were subjected to immunoprecipitation using a FLAG-specific antibody. O-GlcNAcylation of FOXP1 was detected by immunoblotting. *, non-specific signal. ( D ) An in vitro kinase assay reaction mixture consisting of GST-tagged CHK1, GST-tagged FOXP1, and the phosphor group donor ATR-γ-S, with or without CHK1 inhibitor UCN-01, was incubated for 0.5 h, and further incubated with p-Nitrobenzyl mesylate for 2 h. The phosphorylation signals were detected via immunoblotting using a thiophosphate ester-specific antibody. ( E ) In vitro phosphorylation of FOXP1 and its T236A/S396A/S440A mutant by CHK1 was established as described in ( D ). ( F ) In vitro phosphorylation of FOXP1 and its S396A mutant by CHK1 was established as described in ( D ). ( G ) HEK293T cells transfected with FLAG-tagged FOXP1 treated with 2 mM HU for 1 h with or without CHK1 inhibitors, Rabusertib (5 μM) or UCN-01 (50 nM), were subjected to immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were examined via immunoblotting using a specific antibody to detect FOXP1 phosphorylation at S396. ( H ) HEK293T cells overexpressing wild-type FLAG-tagged FOXP1, its phosphorylation deficient mutant S396A, or its phosphorylation mimic mutant S396D were subjected to immunoprecipitation using a FLAG-specific antibody. O-GlcNAcylation of FOXP1 was detected via immunoblotting. ( I ) HEK293T cells overexpressing GFP-tagged OGT and wild-type FLAG-tagged FOXP1, S396A or S396D mutants were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. ( J ) HEK293T cells transfected with wild-type FLAG-tagged FOXP1, S396A, or S396D mutant were treated with 2 mM HU for 1 h and subjected to immunoprecipitation and immunoblotting using the indicated antibodies. .

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Incubation, Phospho-proteomics, Mutagenesis

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) HEK293T cells treated with 2 mM HU for 1 h or untreated were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. ( B ) The potential phosphorylation motif recognized by CHK1 on FOXP1. ( C , D ) In vitro phosphorylation of FOXP1 and its T236A ( C ) or S440A mutant ( D ) by CHK1 were conducted using ATR-γ-S as the phosphor group donor. ( E ) HEK293T cells transfected with FLAG-tagged FOXP1 or its S396A mutant were subjected to immunoprecipitation using a FLAG-specific antibody. The immunoprecipitates were examined via immunoblotting using a specific antibody to detect FOXP1 phosphorylation at S396. ( F ) O-GlcNAcylation stoichiometry of FLAG-tagged FOXP1 S396A or S396D were analyzed and examined via immunoblotting with FLAG-specific antibody. ( G ) Upper panel: Wild-type and S396A or S396D knock-in HEK293 cells were treated with HU for the indicated time before the whole cell lysates were harvested for immunoblotting with the indicated antibodies. Lower panel: genome sequence of wild-type and mutant HEK293 cell lines. ( H ) GST-tagged FOXP1 were subjected to in vitro kinase assay catalyzed by CHK1 or not, followed by incubation with HEK293T cell lysate before GST pulldown. The glutathione bead-bound signals were detected by immunoblotting.

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, In Vitro, Mutagenesis, Transfection, Knock-In, Sequencing, Kinase Assay, Incubation

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) H1975 cells expressing FLAG-tagged FOXP1 or its mutants were subjected to immunostaining using FLAG-specific antibody, and the nuclei were detected with DAPI. ( B ) HEK293T cells transfected with FLAG-tagged FOXP1 or its mutants were treated with 2 mM HU for 1 h before chromatin fractionation. ( C ) HEK293T cells transfected with FLAG-tagged FOXP1 or its deletion mutants were preincubated with Thiamet-G before immunoprecipitation and then immunoblotting using the indicated antibodies. *, non-specific signal. ( D ) HEK293T cells transfected with FLAG-tagged FOXP1 or its deletion mutants and HA-tagged ATRIP were subjected to immunoprecipitation and then immunoblotting using the indicated antibodies. ( E ) FOXP1 knockdown HEK293 cells transfected with wild-type FLAG-FOXP1 or its pathogenic mutants were incubated with 100 nM CPT for 8 h or left untreated before harvested for PI staining and flow cytometric analysis. The percentage of S phase population was analyzed, mean ± SD (biological replicates, n = 3) is shown. **** P < 0.0001, P values were calculated by two-way ANOVA, followed by Sidak’s test. P value: siNC vs siFOXP1, 2.75e-008; siFOXP1 vs siFOXP1 + FOXP1 WT, 1.28e-007; siFOXP1 + FOXP1 WT vs siFOXP1 + R465G, 1.50e-006; siFOXP1 + FOXP1 WT vs siFOXP1 + R514C, 3.66e-006; siFOXP1 + FOXP1 WT vs siFOXP1 + R465T, 6.25e-007; siFOXP1 + FOXP1 WT vs siFOXP1 + R514H, 7.92e-006. ( F ) Statistical analysis of the IdU/CldU ratio mean in Fig. , mean ± SD (biological replicates, n = 3) is shown. ** P < 0.01, *** P < 0.001, P values were calculated by one-way ANOVA, followed by Dunnett’s test. P value: shNC vs shFOXP1, 0.0013, shFOXP1 vs shFOXP1+WT, 0.0008; shFOXP1+WT vs shFOXP1 + R465G, 0.0004; shFOXP1+WT vs shFOXP1 + R514C, 0.0005; shFOXP1+WT vs shFOXP1 + R465T, 0.0004; shFOXP1+WT vs shFOXP1 + R514H, 0.0004. ( G ) Functions of FOXP1 domains identified in this study.

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Expressing, Immunostaining, Transfection, Fractionation, Immunoprecipitation, Western Blot, Knockdown, Incubation, Staining

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) Biotin-labeled ssDNA conjugated on streptavidin magnetic beads were incubated with GST-tagged FOXP1 or its mutants purified from E. coli . Streptavidin bead-bound FOXP1 was detected via immunoblotting using the indicated antibodies. * degraded GST-FOXP1. ( B ) HEK293T cells transfected with wild-type FLAG-FOXP1 or its pathogenic mutants were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. ( C ) HEK293T cells transfected with wild-type FLAG-FOXP1 or its pathogenic mutants, preincubated with or without Thiamet-G, were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. ( D ) HEK293T cells transfected with wild-type FLAG-FOXP1 or its pathogenic mutants were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. ( E ) FOXP1 knockdown HEK293T cells transfected with wild-type FLAG-FOXP1 or its pathogenic mutants were treated with 2 mM HU for 1 h before being lysed for immunoblotting with the indicated antibodies. ( F ) Upper panel: schematic of the DNA fiber assay examining stalled replication fork stability. Middle panel: representative images of CldU and IdU replication tracks. Lower panel: FOXP1 levels in different H1975 cells. ( G ) Statistical analysis of the IdU/CldU ratio of DNA fibers (from F ); the mean IdU/CldU ratio (red line) ±SD is shown. n , DNA fiber number, **** P < 0.0001, P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: shNC vs shFOXP1, 4.15e-015; shFOXP1 vs shFOXP1+WT, 1.93e-024; shFOXP1+WT vs shFOXP1 + R465G, 8.49e-030; shFOXP1+WT vs shFOXP1 + R514C, 4.98e-028; shFOXP1+WT vs shFOXP1 + R465T, 3.76e-018; shFOXP1+WT vs shFOXP1 + R514H, 3.69e-019. .

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Labeling, Magnetic Beads, Incubation, Purification, Western Blot, Transfection, Immunoprecipitation, Knockdown

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: FOXP1 served as a scaffold protein to facilitate the recruitment of ATR and subsequent ATR activation, via directly binding to both RPA-coated ssDNA and the ATR–ATRIP complex. FOXP1 O-GlcNAcylation represses its interaction with ATR, and FOXP1 CHK1-mediated phosphorylation at S396 antagonizes its O-GlcNAcylation under conditions of replication stress.

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Activation Assay, Binding Assay, Phospho-proteomics

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-FOXP1 polyclonal antibody , ABclonal Technology , Cat # A12685.

Techniques: Recombinant, Sequencing, shRNA, Modification, Transfection, Protease Inhibitor, Cloning, In Situ, Software

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet: Computationally identified mesenchymal clusters represent spatially distinct populations (A) Violin plots showing the expression of Hoxb6 or Ptn in each cluster. (B) Fluorescence in situ hybridization for Hoxb6 and Ptn in E 11.5 lungs. Scale bar shows 25 μm. (C) Violin plots showing the expression of Lef1 or Foxp1 in each cluster. (D) E 11.5 lungs immunostained for cluster 0 marker Lef1 or for cluster 1 marker Foxp1 and counterstained with Hoechst. Scale bars show 25 μm. (E) Quantifications of Lef1 and Foxp1 intensity profiles emanating from the epithelium (for Lef1) or from the mesothelium (for Foxp1). Schematics show lines and direction along which intensity profiles were measured. Mean and SD are plotted (n = 4). (F) Schematic depicting the sub-epithelial and sub-mesothelial compartments of the mesenchyme. (G) Heatmap showing the expression of genes specific to either mesenchymal compartment. Genes (rows) are clustered based on the dendrogram to the right. Cells (columns) are clustered based on the dendrogram above, and each column is color-coded according to the original cluster identity from Figure 1 B. (H) UMAP of mesenchymal and smooth muscle cells color-coded according to the sum of their expression of either sub-epithelial or sub-mesothelial mesenchyme marker genes. Dotted line indicates the location of smooth muscle cells

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Expressing, Fluorescence, In Situ Hybridization, Marker

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet: Wnt signaling regulates cell identity in the embryonic pulmonary mesenchyme. (A) Bubble plot showing the enrichment percentage and adjusted p value of relevant GO terms identified based on genes upregulated in each mesenchymal cluster. (B) Heatmap showing the expression of Wnt-associated genes upregulated in either mesenchymal cluster. Activators and targets are colored in blue, inhibitors are colored in red. (C) Heatmap showing the expression of Wnt ligands, secreted inhibitors, and receptors detected in either mesenchymal cluster and in clusters containing cells from the mesothelium (meso), vascular endothelium (ve), epithelium (ep), and smooth muscle (sm). (D–G) Confocal sections and quantification of Lef1 and Foxp1 intensity profiles around branch L.L2 in lungs isolated at E 11.5 from CD1 embryos and immunostained for Lef1 or Foxp1 after treatment with either DMSO, LiCl (10 mM), or IWR1 (100 μM) for 24 h (n = 2–6). Yellow dashed lines indicate the border of the epithelium. Schematics show lines and direction along which intensity profiles were measured. Mean and SEM are plotted, and curves were compared using two-way ANOVA. (H–M) E 12.5 control and Tbx4-rtTA ; tet-O-Cre ; Ctnnb1 fl/fl lungs immunostained for Lef1 or Foxp1 and quantification of Lef1 and Foxp1 intensity profiles (n = 3). Low-magnification z-projections (H and I) and high-magnification confocal slices (J and K) are shown. Scale bars show 50 μm. ∗ indicates p<0.05, ∗∗ indicates p<0.001, and ∗∗∗ indicates p<0.0001

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Expressing, Isolation, Control

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet: Regulators and features of smooth muscle differentiation (A) Sections of E 12.5 Dermo1-Cre/+; Yap fl/fl ; mTmG/+ lungs and littermate controls immunostained for GFP and either Yap1, Lef1, Foxp1, or αSMA. Insets show zoomed-in view of the mesenchyme to highlight the decrease in mesenchymal Yap1 levels in mutants. Yap1 + cells in the mesenchyme of mutants are vascular endothelial cells (ve, indicated by white arrowheads), which are not targeted by Dermo1-Cre . ep is epithelium. Scale bars show 50 μm. (B) Scaled expression of genes involved in cytoskeleton, cell adhesion, and extracellular matrix versus cell loadings along DC1 compared to the expression profiles of the smooth muscle markers Acta2 and Myocd (dotted lines). Pearson correlation coefficients and significance are indicated and lines represent smoothed data with SE shaded in gray. (C) Simplified pathway diagram depicting the steps of proliferative metabolism and showing relevant enzymes at each step. Enzymes that are significantly downregulated along DC1 are indicated in bold red font, with a significance of spline fit indicated by asterisks. ∗ indicates p < 0.05, ∗∗ indicates p < 0.001, and ∗∗∗ indicates p < 0.0001

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Expressing

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Recombinant, RNAscope, Multiplex Assay, Mutagenesis, Software, Sequencing

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: Elevated E2FB level in its own expression domain inhibits cell proliferation in young leaves and disturbs quiescence in older leaves. A, Representative confocal laser scanning microscopy images of the abaxial leaf surface from the first leaf pair of the transgenic line pgE2FB-3×vYFP at 6 and 10 DAG (top), and localization in the epidermis and vascular tissues of the same transgenic line at 10 DAG (bottom). The YFP signal (green) is counterstained for cell membrane with PI (red). Yellow arrows point toward dividing protodermal cells, yellow arrowheads indicate stomatal meristemoids, green arrowheads label fully developed stomata guard cells, blue arrowheads mark elongated pavement cells, and red arrowheads show elongated vascular cells with GFP signal in their nucleus. Scale bars = 20 μm (top) and 25 μm (bottom). B, Images of the wild type (WT) and the transgenic line with high E2FB expression (pgE2FB-GFP line 72) grown for 9 DAG in vitro and for 20 DAG on soil. Scale bars = 0.5 cm. C, Representative images of the abaxial epidermal cell layer of the first leaf pair from wild-type and pgE2FB-GFP line 72 seedlings (12 DAG) taken by differential interference contrast microscopy, for which the imprints were made by the gel casting method. An example of an elongated puzzle-formed pavement cell is outlined in red (left). Arrows indicate straight cell walls inside the cell, whereas arrowheads mark newly formed cell walls inside the elongated pavement cells. Scale bars = 20 μm. D, Quantification of the total number of epidermal cells from the first leaf pair of the wild type and two pgE2FB-GFP transgenic lines (lines 72 and 93). Values represent means and error bars indicate the sd. Significance was determined by Student’s t test; a, P < 0.05. n = 3 and n > 600. The quantifications of cellular parameters are summarized in Supplemental Tables S1 and S2 from 8 and 12 DAG leaves, respectively. Data information, n = biological repeat, n = samples per biological repeat, here and in following figure legends.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Expressing, Confocal Laser Scanning Microscopy, Transgenic Assay, Membrane, In Vitro, Microscopy

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: RBR efficiently counteracts excess E2FB accumulation in proliferating, but not in differentiating, first leaf pairs. A, Relative expression levels of ORC2, CDKB1;1, CYCD3;1, and RBR in the wild type (WT) and pgE2FB-GFP line 72 from the developing first leaf pair of seedlings at 8, 10, 12, and 15 DAG. Values represent the mean of fold change normalized to the value of the relevant transcript of the wild type at 8 DAG, which was set arbitrarily at 1. Error bars indicate the sd. a, P < 0.05; statistical significance determined using Student’s t test between the wild type and the transgenic line at a given time point (n = 3, n > 50). Abbreviations of genes and primer sequences are listed in Supplemental Table S3. B, The phosphorylation level of RBR on the conserved Ser site at position 911 (P-RBRS911) was followed in the developing first leaf pairs of two independent pgE2FB-GFP-expressing lines (lines 93 and 72) with different E2FB protein levels and compared to the wild type at the indicated time points (DAG) using anti-RBR and P-RBRS911-specific antibody (anti-P-Rb807/811) in immunoblot analysis. C, To follow RBR accumulation in conjunction with E2FB level, anti-RBR, anti-E2FB, and anti-GFP antibodies were used in immunoblot analysis of proteins in the developing first leaf pairs in the same transgenic lines as in B. In the top set of blots, the antibody labels RBR (arrow); in the second set, the anti-E2FB antibody labels both the E2FB-GFP (arrow) and the endogenous E2FB (arrowhead); and in the third set, the anti-GFP antibody marks the accumulation of the E2FB-GFP fusion protein (arrow). D, Co-IP of RBR in the E2FB-GFP pull-down was labeled on the immunoblot with anti-RBR. On the same gel, 1/80 of the IP from the extract of the pgE2FB-GFP 72 line was loaded as input. For comparison, 1/20 of IP was loaded for all genotypes in C. Nonspecific membrane-bound proteins stained by Coomassie-blue were used as loading controls (C and D). Note: The relative intensities of the protein bands in B and C are quantified in Supplemental Figure S4, A and B (B) and C and D (C), and the measurements related to proteins in C and D are quantified in Supplemental Figure S4E.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Expressing, Transgenic Assay, Phospho-proteomics, Western Blot, Co-Immunoprecipitation Assay, Labeling, Comparison, Membrane, Staining

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: E2FB restricts cell proliferation in developing first leaf pairs. A and B, Total cell number (A) and ratio of small-sized cells (<60 μm2; B) in the epidermis of the first leaf pairs from the wild type (WT), the e2fb-1 and e2fb-2 mutants, and the e2fb-2 mutant expressing E2FB-GFP under its own promoter (e2fb-2 E2FB-GFP lines 1 and 2) at 12 DAG (n = 3, n > 600). Error bars indicate the sd. a, P < 0.05, statistical significance determined using Student’s t test between the wild type and the two e2fb mutants; b, P < 0.05, statistical significance between the complemented lines and e2fb mutants. C, Comparison of the ORC2, MCM3, CDKB1;1, CYCA2;3, CYCD3;1, and RBR transcript levels in the first leaf pairs of seedlings of the e2fb-2 and e2fb-1 mutants and the wild type at 8, 10, 12, and 15 DAG. Values represent the mean of fold change normalized to the value of the relevant transcript of the wild type at 8 DAG, which was arbitrarily set at 1 (n = 3, n > 50). a, P < 0.05, statistical significance determined using Student’s t test between the wild type and the mutant lines. Error bars indicate the sd. Abbreviations of genes and primer sequences are listed in Supplemental Table S3. D, Endogenous E2FB and transgenic E2FB-GFP proteins were detected in 1-week-old seedlings from the wild type and the two complemented lines [e2fb-2 (E2FB-GFP) lines 1 and 2]. The arrow indicates the position of E2FB, and the arrowhead indicates E2FB-GFP. Nonspecific, cross-reacting proteins are used as loading control.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Mutagenesis, Expressing, Comparison, Transgenic Assay, Control

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: E2FB directly binds to CYCD3;1, CDKB1;1, and RBR promoters. A, Schematic representation of the CYCD3;1, CDKB1;1, and RBR promoters; arrow pairs labeled p1, p2, and p3 indicate the positions of the primer pairs used for qPCR analysis. The position of the canonical E2F elements (white arrowheads) and their distance from the start codon (ATG) are depicted. Primer sequences are listed in Supplemental Table S3. B, ChIP followed by qPCR was carried out on chromatin isolated from complemented e2fb-2 E2FB-GFP seedlings (7 DAG) using polyclonal antirabbit GFP antibody; the graph shows fold enrichment calculated as the ratio of chromatin bound to the numbered section of the CYCD3;1, CDKB1;1, and RBR promoters with or without antibody. Shown is a representative experiment with three biological replicates. a and b, P < 0.01, statistically significant enrichment between the relevant fragment and the neighboring fragments (a) and between the relevant regulatory region and the negative control (Actin2; b) determined by Student’s t test. The values represent the means of three technical replicates. Error bars indicate the sd. The enrichment on the Actin2 promoter was arbitrarily set to 1. The labels p1, p2, and p3 on the x axis refer to the regions indicated in A.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Labeling, Isolation, Negative Control

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: Co-overexpression of E2FB and DPA results in reduced leaf and cell size. A, Representative images of wild-type (WT) and p35S::HA-E2FB/DPAOE (HA-E2FB/DPAOE) seedlings grown in vitro (8 and 12 DAG) and on soil (21 DAG). Scale bars = 0.5 cm at 8 and 12 DAG and 1 cm at 21 DAG. B, Representative confocal microscopy images of PI-stained abaxial leaf surfaces taken from the tip to the base of the first leaf pairs from wild-type and HA-E2FB/DPAOE seedlings (8 and 12 DAG). Scale bars = 20 μm. C, Comparison of E2FB expression levels in the developing first leaf pairs of HA-E2FB/DPAOE and wild-type seedlings at 8, 10, 12, and 15 DAG, where the expression of E2FB was set arbitrarily at 1 at each time point. Values represent fold change. Error bars indicate the sd, referring to technical repeats. The data are from one biological replicate (n < 50), and the transcript level correlates well with the HA-E2FB protein accumulation illustrated in D. D, Detection of protein levels of epitope-tagged (HA-E2FB) and endogenous E2FB, DPA, and CDKB1;1 in the first leaf pairs of wild-type and HA-E2FB/DPAOE seedlings at the indicated time points (DAG) using anti-HA, anti-E2FB, anti-DPA, and anti-CDKB1;1 antibodies. The arrowhead indicates the position of HA-tagged E2FB, whereas arrows indicate endogenous E2FB and CDKB1;1 proteins. The asterisk indicates a nonspecific protein cross reaction with the anti-CDKB1;1 antibody. Nonspecific membrane-bound proteins stained by Coomassie-blue were used as loading control.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Over Expression, In Vitro, Confocal Microscopy, Staining, Comparison, Expressing, Membrane, Control

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: Ectopic E2FB/DPA functions as transcriptional activator on cell cycle genes. A, The expression levels of ORC2, MCM3, CDKB1;1, CYCD3;1, and RBR were determined in wild-type (WT) and HA-E2FB/DPAOE seedlings by RT-qPCR. Developing first leaf pairs were analyzed at each time point, as indicated. Values represent the mean of fold change normalized to values of the relevant transcript from the wild type at 8 DAG, which was set arbitrarily at 1. Error bars indicate the sd; a, P < 0.05, statistical significance between the wild type and the transgenic line at a given time point; b, P < 0.05, significance between two consecutive time points determined using Student’s t test (n = 3, n > 100). Abbreviations of genes and the list of primers used in this study are listed in Supplemental Table S3. B, Protein level of RBR, P-RBRS911, HA-E2FB, and endogenous E2FB in the developing first leaf pairs of wild-type and HA-E2FB/DPAOE seedlings at 8, 9, and 12 DAG detected using anti-RBR, anti-P-RBRS911 (anti-P-Rb807/811), anti-E2FB, and anti-CDKA;1 antibodies in immunoblot assays. Note, the relative intensities of the RBR and P-RBRS911 protein bands are quantified in Supplemental Figure S6, F and G. C and D, Co-IP of HA-E2FB with RBR and DPA proteins in wild-type and HA-E2FB/DPAOE seedlings at 7 DAG (C) and in first leaf pairs at 8 DAG (D). Co-IP of RBR or HA-E2FB proteins with DPA was determined through immunoblot analysis with anti-RBR or anti-E2FB antibodies. One twenty-fifth of the IP from the extract was loaded as input. The asterisk indicates a nonspecific protein cross-reaction with the anti-DPA antibody in the input. In B and D, anti-CDKA;1 antibody was used as control. In C, nonspecific membrane-bound proteins stained by Coomassie-blue were used as loading control. The arrowhead in B indicates HA-E2FB and arrows mark the positions of endogenous E2FB, DPA, and CDKA;1 in B–D, respectively.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay, Western Blot, Co-Immunoprecipitation Assay, Control, Membrane, Staining

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: Coexpression of the mutant HA-E2FBΔRBR with DPA, which is unable to transactivate and bind to RBR, hyperactivates meristematic cell divisions in leaf epidermis. A, Representative images of p35S::HA-E2FBΔRBR/DPA (HA-E2FBΔRBR/DPA), wild-type (WT), and p35S::HA-E2FB/DPA (HA-E2FB/DPAOE) plants grown for 20 d on soil. Scale bar = 1 cm. B, Confocal laser scanning microscopy images of PI-stained abaxial leaf surfaces from the first leaf pairs of wild-type and HA-E2FBΔRBR/DPA seedlings at 10 DAG. The white outline shows a typical puzzle-shaped pavement cell. Arrowheads in both images indicate normally dividing meristemoid cells, whereas white circles illustrate clusters of overproliferated meristemoid cells. Scale bars = 20 μm. C, Total CDK histone H1 kinase activity purified by p13suc1-Sepharose beads is shown and compared to Histone H1 from the first leaf pairs at four different developmental time points (8, 10, 12, and 15 DAG). For comparison, the CDKA;1 protein level is also shown in the same leaf samples. Coomassie-stained nonspecific membrane-bound proteins in the range 50–60 kD were used as loading controls. D, Co-IP of RBR and DPB proteins in the GFP-E2FB∆RBR and GFP-E2FA∆RBR pull-down was labeled with anti-RBR and anti-DPB antibodies. On the same gel, 1/12 of the IP from the extracts of the GFP-E2FB∆RBR and GFP-E2FA∆RBR lines was loaded as input. Arrows point toward the specific proteins. The arrowhead indicates a faster-migrating DPB protein. Molecular weight markers are indicated on the left. E, Expression levels of ORC2, CDKB1;1, CYCD3;1, and RBR were followed in two independent HA-E2FBΔRBR/DPA lines (lines 10 and 1) using RT-qPCR. The developing first leaf pairs were analyzed at each time point, as indicated. Values represent the fold change normalized to values of the relevant transcript from the wild type at 8 DAG, which was set arbitrarily at 1. As the two independent lines show the same tendencies, here, n = 2, n > 50. a, P < 0.05, statistical significance between the wild type and the transgenic line at a given time point determined using Student’s t test.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Mutagenesis, Confocal Laser Scanning Microscopy, Staining, Activity Assay, Purification, Comparison, Membrane, Co-Immunoprecipitation Assay, Labeling, Molecular Weight, Expressing, Quantitative RT-PCR, Transgenic Assay

Journal: Plant Physiology

Article Title: E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development ^{1 [CC-BY]}

doi: 10.1104/pp.19.00212

Figure Lengend Snippet: Model explaining the functions of E2FB during leaf development. E2FB has three different activities, and each is dominant at different leaf developmental stages (A) or in different cell types (B). A, Activator E2FB is in its RBR-free form, characteristic of young leaves consisting of mostly proliferating cells. The young meristematic leaf is a nutrient-rich sink tissue, where E2FB is released from the repression of RBR by the CYCD3;1-regulated RBR kinase in a Suc-dependent manner. E2FB controls the activity of RBR by using CYCD3;1 activity to regulate RBR transcriptional and protein level, as well as phosphorylation status. In leaf cells where the growth-promoting signal is weakened, the protein levels of both E2FB and RBR decrease and RBR becomes more active (less phosphorylated) to bind and inhibit E2FB. This repression is important to establish quiescence in leaf cells committed to differentiation. B, In developing leaves, E2FB also forms a repressor complex with RBR in meristemoid leaf cells to corepress their divisions. How this repression is regulated by upstream signal(s) is hitherto unknown.

Article Snippet: Primary antibodies used in immunoblotting experiments were chicken anti-RBR antibody (1:2,000 dilution; Agrisera), mouse monoclonal anti-PSTAIRE (1:40,000 dilution, CDKA;1 specific; Sigma), rabbit polyclonal antibody anti-CDKB1;1 (1:2,000 dilution; Magyar et al., 2005 ), antiphospho-specific Rb (Ser-807/811) rabbit polyclonal antibody (1:500 dilution; Cell Signaling Tech), and anti-E2FB polyclonal rabbit antibody (1:400 dilution, Magyar et al., 2005 ).

Techniques: Activity Assay, Phospho-proteomics

Journal: Cell Reports

Article Title: Reconstitution of the Human Nigro-striatal Pathway on-a-Chip Reveals OPA1-Dependent Mitochondrial Defects and Loss of Dopaminergic Synapses

doi: 10.1016/j.celrep.2019.11.111

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-FOXP1 , Abcam , AB16645.

Techniques: Recombinant, Virus, Software