Journal: Leukemia

Article Title: GCN5 acetylates and regulates the stability of the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia.

doi: 10.1038/leu.2012.265

Figure Lengend Snippet: Figure 1. Functional interaction of GCN5 with E2A-PBX1. (a) Schematic diagram of E2A, PBX1 and E2A-PBX1. (b) Schematic presenting the binding sites for p300 and STAGA in the E2A AD1. (c) E2A-PBX1, p300-HA and FLAG-GCN5 expression vectors were co-transfected into HEK- 293 cells. E2A-PBX1 proteins were immunoprecipitated from whole-cell extracts using an E2A-PBX1 antibody. The proteins were separated by SDS–PAGE and detected by western blotting using antibodies recognizing E2A-PBX1, FLAG-tag, HA-tag or the acetylated lysines in E2A-PBX1. GAPDH was used as a loading control. The input represents 5% of the whole-cell extracts used in each binding reaction. (d) Coomassie blue- stained SDS-gel showing purified GST-tagged E2A-PBX1 proteins. (e) GST-E2A-PBX1, GST-E2A-PBX1-LDFSmut or GST was immobilized on glutathione-sepharose beads. The beads were incubated with 35S-labeled GCN5 and the bound proteins were analyzed by SDS–PAGE and autoradiography. The input represents 2% of the reticulate lysate with 35S-labeled GCN5 used in each binding reaction.

Article Snippet: Cell lysates were cleared by centrifugation and the proteins were subsequently analyzed by immunoblotting with the following antibodies: E2A-PBX1 (556021; BD Biosciences, Franklin Lakes, NJ, USA), E2A (E12/E47; 554199; BD Biosciences), GCN5 (sc-20698), Wnt16 (sc-271897), Pol II (sc-9001) and Tubulin (sc-5546; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Functional Assay, Binding Assay, Expressing, Transfection, Immunoprecipitation, SDS Page, Western Blot, FLAG-tag, Control, Staining, SDS-Gel, Incubation, Labeling, Autoradiography

Journal: Leukemia

Article Title: GCN5 acetylates and regulates the stability of the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia.

doi: 10.1038/leu.2012.265

Figure Lengend Snippet: Figure 2. GCN5 as a subunit in the STAGA complex interacts with E2A-PBX1. (a) The human STAGA complex was affinity purified from nuclear extracts of a FLAG-SPT3-expressing cell line (lane 2) and control HeLa cells (lane 1). Silver-staining of gradient SDS–PAGE gels confirmed the purity of the purified STAGA complex. (b, d) Immobilized GST fusion proteins (as indicated at the top) were incubated with affinity-purified STAGA (b) or HeLa nuclear extract (d) and the interacting STAGA subunits were identified by immunoblotting. The input represents 30% of the purified STAGA or 5% of nuclear extract used in each binding reaction. (c) GST pull-down assays of GST-E2A-PBX1 or GST bound to glutathione-sepharose beads and the purified STAGA complex. The beads were subjected to the nucleosomal HAT assay; histone acetylation indicated the presence of STAGA. The input represents 30% of the purified STAGA used in each binding reaction. (e) Immobilized GST-E2A- PBX1 and bound STAGA were subjected to crosslinking with dithiobis (succinimidylpropionate) and crosslinking of the STAGA subunits GCN5, TAF31 and SPT3 was monitored by immunoblotting. The input represents 30% of the purified STAGA or 5% of nuclear extract used in each binding reaction.

Article Snippet: Cell lysates were cleared by centrifugation and the proteins were subsequently analyzed by immunoblotting with the following antibodies: E2A-PBX1 (556021; BD Biosciences, Franklin Lakes, NJ, USA), E2A (E12/E47; 554199; BD Biosciences), GCN5 (sc-20698), Wnt16 (sc-271897), Pol II (sc-9001) and Tubulin (sc-5546; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Control, Silver Staining, SDS Page, Incubation, Western Blot, Binding Assay, HAT Assay

Journal: Leukemia

Article Title: GCN5 acetylates and regulates the stability of the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia.

doi: 10.1038/leu.2012.265

Figure Lengend Snippet: Figure 3. The GCN5 inhibitor MB-3 reduces acetylation and induces degradation of E2A-PBX1. (a) HEK-293 cells were co-transfected with plasmids expressing E2A-PBX1 and FLAG-GCN5, and treated for 16 h with MB-3 as indicated. E2A-PBX1 protein was immunoprecipitated from whole-cell extracts and acetylation of E2A-PBX1 was monitored by immunoblot using an antibody recognizing acetylated lysines. The input represents 5% of the whole-cell extract used in each binding reaction. (b) The amount of acetylated E2A-PBX1 was determined by densitometric analysis of the scanned films, and expressed relative to the total levels of E2A-PBX1 in the IP fraction using Image J software. The data were derived from the experiment in (a). (c) RCH-ACV, 697 and REH cells were treated with the GCN5 inhibitor MB-3 for 16 h as indicated. E2A-PBX1 protein was immunoprecipitated from whole-cell extracts and acetylation of endogenous E2A-PBX1 was monitored by immunoblot using an antibody recognizing acetylated lysines. REH cells, which do not express E2A-PBX1, were used as a negative control. The input represents 5% of the whole-cell extracts used in each binding reaction. (d) RCH-ACV cells were treated for 14 h with 0, 50 or 100 mM MB-3 and the whole-cell extracts were analyzed by immunoblotting with the indicated antibodies. (e) Leukemic cells from a patient carrying the t(1;19) translocation were isolated, treated with 50 mM MB-3 for 14 h, then analyzed by western blotting using the indicated antibodies. (f) RCH- ACV cells were pre-treated with 100 mM MB-3 for 2 h and protein synthesis was inhibited with cycloheximide at the times indicated. Degradation of E2A-PBX1 protein was monitored by immunoblotting and the bands were quantified using Image J software. The data in the plots are derived from six independent experiments.

Article Snippet: Cell lysates were cleared by centrifugation and the proteins were subsequently analyzed by immunoblotting with the following antibodies: E2A-PBX1 (556021; BD Biosciences, Franklin Lakes, NJ, USA), E2A (E12/E47; 554199; BD Biosciences), GCN5 (sc-20698), Wnt16 (sc-271897), Pol II (sc-9001) and Tubulin (sc-5546; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Binding Assay, Software, Derivative Assay, Negative Control, Translocation Assay, Isolation

Journal: Nature communications

Article Title: The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry.

doi: 10.1038/s41467-017-00593-6

Figure Lengend Snippet: Fig. 7 Kat2a but not Ep300 acts as a coactivator for retinoic acid signaling. a–e RARE-Luciferase activity from NIH3T3 cells treated or not with 1 µM RA for 20 h. a Cells transfected with expression plasmids containing Ep300, Hdac1, or both (n = 4). b Cells transfected with expression plasmids containing human Hdac1 (H1-WT) or Hdac1 mutant (H1-6R) (n = 4). c Cells transfected with expression plasmids containing Rarα, Rarα, and Ep300 or Rarα and Kat2a (n = 3). d Cells transfected with expression plasmids containing Rere, Rere and Ep300 or Rere and Kat2a (n = 3). e Cells treated either with siRNA for Rere, Ep300, or Kat2a (n = 4). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test. f, g RARE-LacZ expression in wild-type Ep300+/+ f and Ep300–/– g embryos at E8.75-E9.0 (dorsal views). h, i In situ hybridization showing somites labeled with Uncx4.1 in wild-type Ep300+/+ h and Ep300–/– i embryos at E8.75-E9.0 (dorsal views). For each genotype at least 5–10 embryos were analyzed. Bar = 100 microns

Article Snippet: Human Ep300 in pCMVb was a gift from Richard Eckner and mouse Kat2a (Gcn5) in pCMV-sport247 (Invitrogen) (Addgene plasmid 23098) was a gift from Sharon Dent.

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Mutagenesis, Two Tailed Test, In Situ Hybridization, Labeling

Journal: Cell reports

Article Title: Heat shock factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat shock response

doi: 10.1016/j.celrep.2023.112557

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant FLAG-GCN5 proteins , Active Motif , Cat#31591.

Techniques: Control, In Situ, Virus, Recombinant, Protease Inhibitor, Transfection, cDNA Synthesis, SYBR Green Assay, Luciferase, Chromatin Immunoprecipitation, Quantitation Assay, Western Blot, Microscopy, Mutagenesis, shRNA, Plasmid Preparation, Software, Quantitative Proteomics, Magnetic Beads, Blocking Assay, Membrane

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Interaction Proteomics of Polycystins 1 and 2 Reveal a Novel Role for the BLOC-1/BORC Lysosomal Positioning Complex

doi: 10.1016/j.mcpro.2025.101091

Figure Lengend Snippet: Validation of PC1-BLOC-1/BORC interaction. A , coimmunoprecipitation of transiently transfected GFP-tagged BLOC-1 components (BLOC1S4, BLOC1S2, and SNAPIN) in cell lines stably expressing PC1-BF ( left panel ) or negative control CD16.7-BF ( right panel ). Cell lysates were incubated with anti-GFP beads and processed for western blotting with anti-FLAG antibodies. The FLAG epitope is present on both PC1-BF (∼90 kDa) and CD16.7-BF (∼75 kDa) constructs (see schematic in A ). B , left panel shows representative pseudocolor merge micrographs of NHPTK RECs fixed and processed for a proximity ligation assay (PLA), depicting amplified fluorescence in the presence of interacting partners (red channel) and nuclei (DAPI). PLA was performed with background controls (primary only and secondary only), a negative control (PC1 + LaminB1), and a positive control (ɣ-tubulin + CEP192) and used to detect PC1+BLOC1S1 and PC1+SNAPIN association using endogenous antibodies. Right panel plot shows a representative quantification of the per-cell amplified PLA signal ( solid black dots ; n = 50 cells) under each condition, with the mean of the population denoted by a horizontal line. ∗∗ denotes p < 0.01 by Student’s t test between the specified population and the PC1 + LaminB1 negative control. The experiment was repeated three times with the same trends. The scale bar represents 20 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Polyclonal antibodies used here were raised against BLOC1S1 (Proteintech; 1:500), BLOC1S2 (Novus; 1:100), DTNBP1 (Cusabio; 1:100), SNAPIN (Proteintech; 1:100), BORCS7 (Cusabio; 1:200), ARL13B (Proteintech; 1:500) and TCTN3 (Cusabio; 1:200).

Techniques: Biomarker Discovery, Transfection, Stable Transfection, Expressing, Negative Control, Incubation, Western Blot, FLAG-tag, Construct, Proximity Ligation Assay, Amplification, Fluorescence, Positive Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Interaction Proteomics of Polycystins 1 and 2 Reveal a Novel Role for the BLOC-1/BORC Lysosomal Positioning Complex

doi: 10.1016/j.mcpro.2025.101091

Figure Lengend Snippet: Functional significance of BLOC-1/BORC. ( A , left series ) Representative pseudocolored merge micrographs of immunofluorescence labeling in ciliated NHPTK RECs showing nuclear staining (DAPI), a cilia marker (ARL13B) and GFP-BLOC-1 protein fusions (BLOC1S4, BLOC1S2, DTNBP1, and SNAPIN). Cilia marked by ARL13B were magnified ( white box ) and shown in zoomed panels. The scale bar represents 10 μm, zoomed panels are 2.5x. ( A , right series ) Pseudocolored merge micrographs of ciliated NHPTK RECs immunolabeled for ciliary markers (Acetylated tubulin, AcTub; TCTN3, a transition zone marker; ARL13B) in parallel with anti-PC1 ( left panel ), or GFP-BLOC1S2 and PC1 ( middle panel ), or GFP-BLOC1S4/PC1 ( right panel ). The cropped insets in the middle and right panels highlight the signal per channel in the basal body region of the cilium. The scale bar represents 10 μm. B , main panel shows representative micrographs of anti-PC1 and anti-ARL13B labeling upon depletion of BLOC-1 components in NHPTK RECs (siBLOC1S2/4 or shBLOC1S1) or knockout in RPE cells (SNAPIN KO), as compared to their respective wild-type (WT) controls. Scale is the same as in ( A ). Corresponding violin plot of the percentage of ciliated cells where PC1 was no longer present at the base of the cilium marked by ARL13B in each condition ( n = 100 cells pooled from three experiments). Values are normalized to the WT control and are statistically different from control ( p < 0.05 by Student’s t test). Solid horizontal lines in each condition denote the median, while dotted lines denote quartiles. C , representative pseudocolor merge micrographs of NHPTK cells processed for endogenous antibody immunolabeling of PC1, lysosomes (as marked by DQ-BSA), together with endogenous anti-SNAPIN ( left panel), anti-BLOC1S1 (middle panel), and anti-BLOC1S2 ( right panel). The dashed white ellipse overlay represents the approximate location of the nucleus in each cell. The boxed white region is zoomed 4x in the insets and individual fluorescence channels are shown to highlight the overlap. The scale bar represents 15 μm. D , left panel shows representative psuedocolor merge micrographs of live-imaged NHPTK RECs loaded with nuclear (Hoechst) and lysosomal (LysoTracker) marker probes after transfection with siRNAs against BLOC1S2, SNAPIN, or PKD1, as compared to mock transfected control (as labeled). The scale bar represents 20 μm. Right panel shows a representative quantification scatter plot of the fraction integrated intensity (of total per cell) corresponding to a 30-pixel wide ring outside of the nuclear mask ( dark blue area) as defined in the cartoon ( light blue area), that was measured ( n > 35 cells) in each condition. Horizontal solid lines denote mean ± SD, and all siRNA treated conditions were significantly different from the mock transfected control ( p < 0.01 by Student’s t test). The experiment was repeated thrice with similar trends. DAPI, 4′,6-diamidino-2-phenylindole; DQ-BSA, dye-quenched bovine serum albumin; PC1, polycystin 1 NHPTK, normal human proximal tubule kidney;; PKD, polycystic kidney disease; REC, renal epithelial cell; RPE, retinal pigment epithelium.

Article Snippet: Polyclonal antibodies used here were raised against BLOC1S1 (Proteintech; 1:500), BLOC1S2 (Novus; 1:100), DTNBP1 (Cusabio; 1:100), SNAPIN (Proteintech; 1:100), BORCS7 (Cusabio; 1:200), ARL13B (Proteintech; 1:500) and TCTN3 (Cusabio; 1:200).

Techniques: Functional Assay, Immunofluorescence, Labeling, Staining, Marker, Immunolabeling, Knock-Out, Control, Fluorescence, Transfection

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Interaction Proteomics of Polycystins 1 and 2 Reveal a Novel Role for the BLOC-1/BORC Lysosomal Positioning Complex

doi: 10.1016/j.mcpro.2025.101091

Figure Lengend Snippet: Role of BLOC-1/BORC in cellular disease models of ADPKD. A , representative psuedocolor merge micrographs of immunofluorescence labeling in NHPTK ( left panels ) and ADPKD Q4004X ( right panels ) RECs showing nuclear (DAPI), and anti-BLOC1S1, -BLOC1S2, -SNAPIN and -BORCS7 labeling. An inverted gray scale image of the antibody channel is shown alongside for easier visualization of the BLOC-1/BORC subunit distribution. The cell and nuclear outlines for each cell are depicted with black dashed lines . The scale bar represents 15 μm. B , Top left : cartoon flowchart of the 3D cystogenic assay in mIMCD-3 cells. Top right : representative micrographs of phase contrast brightfield images of mIMCD-3 control (shScrambled KD) and shRNA knockdown cells seeded in Matrigel, which exhibited cyst growth over a 6-day period. The scale bar represents 150 μm. The number of cysts formed at day 4 ( left lower panel ), and cyst areas ( right lower panel ) were quantified for each knockdown condition. Cyst numbers depicted as symbols, were counted for each condition (mean is shown as dashed horizontal line for each condition), and three replicate experiments are plotted. All mean values were significantly different from the control ( p < 0.01 by Student’s t test). For cyst area, 15 cysts were randomly sampled from the pooled experiments and measured (see ). C , the cyst assay in ( B ) was repeated with control (shScrambled KD) or BLOC-1/BORC shRNA knockdown cells, additionally with or without stably transfected PC1-CTT (see ). Cells were then plated as in ( B ) and the number of cysts quantified ( left panel ) and representative micrographs were taken ( right panel ). The scale bar represents 45 μm. ADPKD, autosomal dominant polycystic kidney disease; BORC, BLOC-one-related-complex; CTT, C-terminal tail; DAPI, 4′,6-diamidino-2-phenylindole; mIMCD, mouse inner medullary collecting duct; NHPTK, normal human proximal tubule kidney; PC1, polycystin 1; REC, renal epithelial cell.

Article Snippet: Polyclonal antibodies used here were raised against BLOC1S1 (Proteintech; 1:500), BLOC1S2 (Novus; 1:100), DTNBP1 (Cusabio; 1:100), SNAPIN (Proteintech; 1:100), BORCS7 (Cusabio; 1:200), ARL13B (Proteintech; 1:500) and TCTN3 (Cusabio; 1:200).

Techniques: Immunofluorescence, Labeling, Control, shRNA, Knockdown, Stable Transfection, Transfection