anti bin2  (Proteintech)

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: Increased BIN2 activity leads to early formation of large vacuoles at stage FG1. (A–J) Confocal laser scanning microscopy images of developing FGs at stage FG1 in wild-type (A), bin2-1 (B–D), and ProSTK:BIN2*-GFP transgenic lines #31 (E–G) and #32 (H–J). Normal FGs are shown in panels (A), (B), (E), and (H). Abnormal FGs with apparent vacuoles are shown in panels (C), (F), and (I). Abnormal FGs with degenerated nuclei are shown in panels (D), (G), and (J). (K) Normal FG at stage FG4 in wild-type. (L) Abnormal FG with one nucleus containing obvious vacuoles at stage FG4 in bin2-1 . (M) Mature FG at stage FG7 in wild-type. (N–P) Aborted FGs in bin2-1 (N), #31 (O), and #32 lines (P) at stage FG7. (Q) Percentages of abnormal FGs at stage FG1 in wild-type, bin2-1 , #31 and #32 lines (R) Percentages of FGs containing vacuoles at stage FG1 in bin2-1 , #31 and #32 lines following 10 µM bikinin and 10 mM LiCl treatments. FGs from three independent pistils were analyzed. (S) Percentages of abnormal FGs at stage FG7. Numbers indicate how many FGs were observed for calculating the ratio in (Q and S). FGs are outlined with dotted lines (A–C, E–F, H–I, and K–M). N represents the nuclei (A–C, E–F, H–I, K–L). DM represents the degenerating megaspores (A–J). AFG represents aborted FGs (N–P). CCN, ECN and SCN indicate the nuclei of central, egg, and synergid cells, respectively (M). FG, female gametophyte. V, vacuole. DFG, degenerated female gametophyte. FG1V, vacuoles formed at stage FG1. Error bars represent means ± SD. Statistically significant differences are tested by one-way ANOVA (** P < 0.01, ). Bars = 10 µm.

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: Activity Assay, Confocal Laser Scanning Microscopy, Transgenic Assay

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: BIN2 is required for female gametophyte development. (A–I) Phenotypic analysis of FG development in the bin2-3 bil1 bil2 triple mutant. (A) Wild-type FG at stage FG1 (Ws ecotype, the wild-type control for bin2-3 bil1 bil2 ). (B) bin2-3 bil1 bil2 FG at stage FG1. (C–D) Mature (C) and aborted (D) FGs at stage FG7 in wild-type plants. (E–H) Images of bin2-3 bil1 bil2 mutant at stage FG7 showing normal FGs (E), degenerated FGs (F), FGs arrested at stage FG1 (G), and FGs arrested at stage FG2 (H). (I) Percentages of abnormal FGs at stage FG7 in wild-type and bin2-3 bil1 bil2 plants. Numbers represent how many FGs were detected for calculating the percentages. Dashed lines outline the FGs (A–C, E). N indicates the FG nuclei (A–B). CCN, ECN and SCN indicate the nuclei of central, egg, and synergid cells, respectively (C, E). DFG represents the degenerated FG exhibiting by their strong autofluorescence (D, F). AN represents the arrested nuclei at stage FG1 (G) or FG2 (H). (J–K) BIN2 localizes in a punctate pattern in FG at stage FG1 (J) and FG7 (K) revealed by immunolocalization assay, and partially co-localizes with the vesicle and tonoplast-expressed marker GFP-VAMP711. White dashed lines outline the FGs. White arrows indicate that BIN2 co-localizes with GFP-VAMP711. Bars = 10 µm.

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: Mutagenesis, Control, Marker

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: Early formation of large vacuoles impairs the female gametophyte development. (A–F) Expression of the vesicle and tonoplast-expressed marker ProUBQ10:GFP-VAMP711 in wild-type (A–C) and bin2-1 (D–F) FGs at the early (A, D), middle (B, E), and late (C, F) stages of FG1. Nuclei were stained with H33258. OIn and IIn indicate the outer and inner integuments, respectively. White dotted lines outline the FGs. Bars = 10 µm.

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: Expressing, Marker, Staining

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: BIN2 interacts with VLG in vivo and in vitro. (A) Yeast two-hybrid assay indicates the interaction between BIN2 and VLG. Protein interactions are evaluated on YPD medium lacking Leu and Trp (left) or Leu, Trp, and His (right). (B) BiFC assay reveals the interaction between BIN2 and VLG. Bars = 50 µm. (C) In vitro pull-down assay indicates the direct interaction between GST-BIN2 and MBP-VLG. (D) In vivo co-immunoprecipitation assay reveals the interaction between BIN2-MYC and VLG-GFP.

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: In Vivo, In Vitro, Y2H Assay, Bimolecular Fluorescence Complementation Assay, Pull Down Assay, Co-Immunoprecipitation Assay

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: BIN2 regulates VLG stability. (A–B) VLG is more stable in bin2-1 than wild-type and bin2-3 bil1 bil2 . Representative immunoblot images (A), and statistical analysis of relative VLG-GFP protein levels (B). Total proteins were extracted from wild-type (Col-0 and Ws), bin2-1 and bin2-3 bil1 bil2 plants expressing Pro35S:VLG-GFP , respectively. VLG was detected using an anti-GFP antibody, and tubulin was used as an internal control. The integrated optical density (IOD) values of VLG and tubulin were quantified using Image Lab software (Bio-Rad). The relative protein levels of VLG were calculated by IOD VLG /IOD tubulin . Data represent means ± SD of three independent experiments. (C) Significantly reduced numbers of vacuole-containing FGs at stage FG1 in ProFM1:VLG-RNAi bin2-1 plants. FGs from four independent pistils were analyzed for each line. (D) Significantly increased numbers of vacuole-containing FGs at stage FG1 in ProFM1:VLG-GFP transgenic plants. FGs from two independent pistils were examined for each line. (E) Percentages of abnormal FGs at stage FG7. #1 and #2 represent two independent lines expressing ProVLG:VLG-GFP in bin2-3 bil1 bil2 mutant. FGs from 10 individual pistils were examined for each plant. (F) MG132 suppresses VLG degradation in a cell-free degradation assay. A mixture of MBP-VLG with wild-type protein extracts was incubated with mock (DMSO) or 100 µM MG132 for the indicated times, and was detected using an anti-MBP antibody. (G) VLG degradation was attenuated in bin2-1 in a cell-free degradation assay. MBP-VLG was mixed with equal amounts of total protein extracts of wild-type and bin2-1 , respectively. The mixture was incubated for the indicated times and detected with anti-MBP antibody. Dots indicate the degenerated MBP-VLG (F–G). (H–I) VLG was more stable in bin2-1 than that in wild-type plants under 100 µM CHX treatment at indicated time. Representative immunoblot images (H) and statistical analysis (I). The relative abundance of VLG-GFP protein was analyzed as described for (A–B) and compared with IOD VLG /IOD tubulin at 0 h. Data represent means ± SD of three independent experiments. Asterisks indicate the significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA, ).

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: Western Blot, Expressing, Control, Software, Transgenic Assay, Mutagenesis, Degradation Assay, Incubation

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: BIN2 increases the stability of VLG via phosphorylation. (A) Predicted structure of VLG protein by Alphafold 2 ( https://www.alphafold.ebi.ac.UK ). The arrows indicate the phosphorylated residues determined by mass spectrometry. (B) The translated open reading frame of VLG. The black box represents the protein-coding exon; thin lines denote untranslated regions. Arrows indicate the positions of the phosphorylated residues. C, BIN2 phosphorylates VLG. The phosphorylation of the phospho-defective protein MBP-VLG S/T-A by BIN2 was significantly decreased and attenuates its stability compared with wild-type VLG and the phospho-mimic protein MBP-VLG S/T-D in vitro. MBP, MBP-BIN2, MBP-VLG, and phosphorylation site-mutated MBP-VLG were immunoprecipitated and purified using anti-MBP-conjugated beads. Different combinations of purified proteins were mixed and incubated at 30°C. (D) VLG-GFP was less abundant following CIP treatment, but more abundant following GSK3β treatment in vitro. It was immunoprecipitated using anti-GFP-conjugated beads from Pro35S:VLG-GFP transgenic plants in wild-type, after which the beads were incubated with or without CIP or GSK3β. (E) The mutant protein MBP-VLG S/T-A was less stable while MBP-VLG S/T-D was more stable than wild-type MBP-VLG in vitro. MBP-VLG, MBP-VLG S/T-A , and MBP-VLG S/T-D proteins were mixed with the wild-type protein extracts for the indicated times, respectively, and detected using an anti-MBP antibody. Dots indicate the degenerated MBP-VLG.

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: Phospho-proteomics, Mass Spectrometry, In Vitro, Immunoprecipitation, Purification, Incubation, Transgenic Assay, Mutagenesis

Journal: The Plant Cell

Article Title: Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

doi: 10.1093/plcell/koad007

Figure Lengend Snippet: Proposed model for how the BIN2-VLG module influences female gametophyte development by regulating vacuole formation at an early developmental stage. (A) In wild-type plants, the accumulation and degradation of VLG is usually in balance. There are no visible large vacuoles in FG at stage FG1. (B) In bin2-1 , increased BIN2 activity enhances VLG stability through preventing its degradation by the 26S proteasome via phosphorylating VLG. This promotes large vacuole formation in FG at stage FG1, resulting in defective FG development. (C) In the bin2-3 bil1 bil2 triple mutant, decreased BIN2 activity reduces VLG stability, leading to smaller vacuoles in FGs at stage FG2, causing defective FG development. Taken together, our study revealed that BIN2 stabilizes VLG by interacting and phosphorylating VLG, and the correct spatiotemporal formation of vacuoles regulated by BIN2-VLG module is required for normal FG development. The arrow and fork represent the enhanced interaction and the loss of interactions between BIN2 and VLG, respectively. P indicates protein phosphorylation.

Article Snippet: The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in at 4°C overnight.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics

Journal: Cell Proliferation

Article Title: Identifying the key genes of Epstein–Barr virus‐regulated tumour immune microenvironment of gastric carcinomas

doi: 10.1111/cpr.13373

Figure Lengend Snippet: The association between distribution and expression of key hub genes and cell subsets in gastric cancer. The scatter plots showed the association between CD8+/CD4+ T cells and GBP1 (A), IRF1 (C) and LAP3 (E). The scatter plots showed the association between macrophage–monocyte/myeloid dendritic cells and BIN2 (B), ITGAL (D) and LILRB1(F). The cell subset profiles (UMAP) of GSE134520 (G) and GSE167297 (N) were given. The expression of GBP1 (H and O), BIN2 (I and P), IRF1 (J and Q), ITGAL (K and R), LAP3 (L and S) and LILRB1(M and T) in cell subsets were presented.

Article Snippet: The microarrays were then quenched with 3% hydrogen peroxide and blocked with 10% goat serum for 30 min. Then, the sections were incubated overnight with primary antibody for GBP1 (Santa Cruz, sc‐53857, 1:50), BIN2 (Santa Cruz, sc‐376391, 1:50) and LAP3 (Santa Cruz, sc‐376270, 1:50) at 4 °C.

Techniques: Expressing

Journal: Cell Proliferation

Article Title: Identifying the key genes of Epstein–Barr virus‐regulated tumour immune microenvironment of gastric carcinomas

doi: 10.1111/cpr.13373

Figure Lengend Snippet: Immune‐related pathways could be activated by each of the six key hub genes in gastric cancer. The TCGA‐STAD expression dataset was used, and the cutoff values were set at 35%. The gene set enrichment analysis of GBP1 (A), BIN2 (B), IRF1 (C), ITGAL (D), LAP3 (E) and LILRB1(F)

Article Snippet: The microarrays were then quenched with 3% hydrogen peroxide and blocked with 10% goat serum for 30 min. Then, the sections were incubated overnight with primary antibody for GBP1 (Santa Cruz, sc‐53857, 1:50), BIN2 (Santa Cruz, sc‐376391, 1:50) and LAP3 (Santa Cruz, sc‐376270, 1:50) at 4 °C.

Techniques: Expressing

Journal: Cell Proliferation

Article Title: Identifying the key genes of Epstein–Barr virus‐regulated tumour immune microenvironment of gastric carcinomas

doi: 10.1111/cpr.13373

Figure Lengend Snippet: Expression of key hub genes and regulation of latent/lytic proteins of EBV expression in gastric tissue. Immunohistochemistry assays were performed to detect expression and distribution of GBP1, BIN2 and LAP3 proteins in gastric tissue microarray (A). Quantitative analysis results are presented (B–D). The pSG5‐EBNA1, pSG5‐LMP1, pSG5‐BMRF1, pSG5‐BGLF4 and pcDNA3.1‐LMP2A were transfected into MGC‐803 cells. Then, the mRNA levels were determined by the real‐time PCR method. The expression of associated gene was normalized to GAPDH by using a △△GA method (E). * p < 0.05; ** p < 0.01.

Article Snippet: The microarrays were then quenched with 3% hydrogen peroxide and blocked with 10% goat serum for 30 min. Then, the sections were incubated overnight with primary antibody for GBP1 (Santa Cruz, sc‐53857, 1:50), BIN2 (Santa Cruz, sc‐376391, 1:50) and LAP3 (Santa Cruz, sc‐376270, 1:50) at 4 °C.

Techniques: Expressing, Immunohistochemistry, Microarray, Transfection, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation

doi: 10.1172/JCI136457

Figure Lengend Snippet: (A) Western blot analysis of BIN2 expression in human tissue lysates; GAPDH was used as loading control. (B) IP of BIN2 was performed with human platelet lysates using an anti-BIN2 antibody. BIN2, STIM1, and RhoA were detected by Western blot. Representative result of 3 independent experiments. Lane 4 was run on the same gel as 1 to 3, but the lanes were noncontiguous. (C) Recombinant GST-tagged STIM1 C-tail incubated with recombinant BIN2 protein. Pulldown of the GST-tagged STIM1 was performed, and the resulting fractions were analyzed by SDS page and silver staining. (D) Western blot analysis of BIN2 expression in the indicated tissue lysates of WT and Bin2–/– mice with GAPDH as loading control. See complete unedited blots in the supplemental material.

Article Snippet: Untreated or activated platelets were lysed at the respective time points, separated by SDS-PAGE, and blotted on a PVDF membrane, on which the proteins were detected using anti-BIN2 antibody (Proteintech, 14245-1-AB), anti-STIM1 antibody (Cell Signaling Technology, 4916), anti-IP 3 R antibody (Merck-Millipore, 07-1210), anti-RhoA (Cytoskeleton, ARH04), and anti-GAPDH (MilliporeSigma, G9545).

Techniques: Western Blot, Expressing, Control, Recombinant, Incubation, SDS Page, Silver Staining

Journal: The Journal of Clinical Investigation

Article Title: BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation

doi: 10.1172/JCI136457

Figure Lengend Snippet: (A) Western blot analysis of BIN2 expression in WT and Bin2fl/fl,Pf4Cre platelet lysates with GAPDH as loading control. (B) Ca2+ store content was measured upon stimulation with 5 μM ionomycin in the presence of 0.5 mM EGTA (n = 10). (C) Ca2+ store release was measured in the absence of extracellular Ca2+ upon treatment with 0.1 μM or 5 μM thapsigargin (TG) (n ≥ 8) and (D) Ca2+ influx upon treatment with 0.1 μM TG (n = 8). (E and F) Representative traces and statistical analysis of store release (E) and Ca2+ influx (F) upon activation of the platelets with the indicated agonists (n ≥ 8). (G) Western blot analysis of WT and Bin2fl/fl,Pf4Cre platelet lysates detecting the expression of STIM1 with GAPDH as loading control. Values are depicted as mean ± SD, and P values were calculated using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001. See complete unedited blots in the supplemental material.

Article Snippet: Untreated or activated platelets were lysed at the respective time points, separated by SDS-PAGE, and blotted on a PVDF membrane, on which the proteins were detected using anti-BIN2 antibody (Proteintech, 14245-1-AB), anti-STIM1 antibody (Cell Signaling Technology, 4916), anti-IP 3 R antibody (Merck-Millipore, 07-1210), anti-RhoA (Cytoskeleton, ARH04), and anti-GAPDH (MilliporeSigma, G9545).

Techniques: Western Blot, Expressing, Control, Activation Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation

doi: 10.1172/JCI136457

Figure Lengend Snippet: (A and B) Determination of whole-cell tyrosine phosphorylation pattern in WT and Bin2fl/fl,Pf4Cre platelets upon activation with convulxin (CVX) (A) or rhodocytin (RC) (B). The samples were taken at the indicated time points, lysed, and Western blot analysis was performed with the indicated phospho-specific and pan-antibodies. This blot is representative of 3 independent experiments. (C) Quantification of inositol monophosphate (IP1), a specific metabolite of inositol-1,4,5-trisphosphate (IP3), produced upon activation with the indicated agonist (n = 3). Representative results of 3 independent experiments. (D) Western blot analysis of IP3R expression in WT and Bin2fl/fl,Pf4Cre platelet lysates; GAPDH was used as loading control. (E and F) Ca2+ concentrations in the cytoplasm of WT and Bin2fl/fl,Pf4Cre platelets upon treatment with UV light–inducible IP3 in the (E) absence or (F) presence of extracellular Ca2+ (n = 8). (G) Bin2fl/fl,Pf4Cre platelet lysates were incubated with recombinant BIN2-HIS protein, followed by a purification step with NI-NTA beads. The different fractions were eluted and analyzed by Western blotting using BIN2-, STIM1-, and IP3R-specific antibodies. Representative result of 3 independent experiments. Values are depicted as mean ± SD, and P values were calculated using the Mann-Whitney U test. **P < 0.01. See complete unedited blots in the supplemental material.

Article Snippet: Untreated or activated platelets were lysed at the respective time points, separated by SDS-PAGE, and blotted on a PVDF membrane, on which the proteins were detected using anti-BIN2 antibody (Proteintech, 14245-1-AB), anti-STIM1 antibody (Cell Signaling Technology, 4916), anti-IP 3 R antibody (Merck-Millipore, 07-1210), anti-RhoA (Cytoskeleton, ARH04), and anti-GAPDH (MilliporeSigma, G9545).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Produced, Expressing, Control, Incubation, Recombinant, Purification, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation

doi: 10.1172/JCI136457

Figure Lengend Snippet: dSTORM images of (A) resting mouse platelets on glycine or (B) thrombin-activated WT and Bin2fl/fl,Pf4Cre mouse platelets on fibrinogen, stained for BIN2. Scale bars 5 μm (upper panel) and 2 μm (lower panel). Dual-color dSTORM allows resolution of the distribution of STIM1 (magenta) and BIN2 (green) in (C) resting and (D) activated human platelets; dashed white lines indicate areas of neighbor density analysis (E) between STIM1 (magenta) and BIN2 (green), with discrete ring areas in which the BIN2 density was determined (dark gray). In activated spread platelets, only localizations in the inner region where the organelles and intercellular membrane compartments accumulate were considered for STIM1. Colocalization hotspots of STIM1 and BIN2 are identified by analyzing the BIN2 density at increasing radial distances to STIM1: Histograms of the distance-dependent BIN2 density peak maxima found for (F) resting (n = 6, Supplemental Figure 7A) and (G) activated human platelets (n = 14, Supplemental Figure 7B). The distinct STIM1 to BIN2 distance regions with increased accumulation of colocalization hotspots are highlighted in light gray. Scale bars (C and D): 2 μm.

Article Snippet: Untreated or activated platelets were lysed at the respective time points, separated by SDS-PAGE, and blotted on a PVDF membrane, on which the proteins were detected using anti-BIN2 antibody (Proteintech, 14245-1-AB), anti-STIM1 antibody (Cell Signaling Technology, 4916), anti-IP 3 R antibody (Merck-Millipore, 07-1210), anti-RhoA (Cytoskeleton, ARH04), and anti-GAPDH (MilliporeSigma, G9545).

Techniques: Staining, Membrane