Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: AC021683.2 knockdown accelerates ubiquitination and degradation of BCLAF1 in HL60-ADR cells under treatment with Ara-C (A) RT-qPCR was used to detect the subcellular fractions of AC021683.2 in HL60-ADR cells. U6 and β-actin were used as markers of the nucleus and cytoplasm, respectively. (B) Silver staining of AC021683.2 RNA pull-down proteins. A probe non-specific for AC021683.2 was used as control. Mass spectrometry analysis identified BCLAF1 (red arrow) as a protein that interacts with AC021683.2 . (C) AC021683.2 RNA pull-down followed by western blot validated the interaction with BCLAF1. (D) RT-qPCR analysis of BCLAF1 mRNA levels in control and AC021683.2 knockdown HL60-ADR cells in the presence of 5 μM Ara-C for 48 h (Data are represented as mean ± S.D. from triplicate experiments). (E) Western blotting analysis of BCLAF1 protein levels in control or AC021683.2 -knockdown HL60-ADR cells treated with or without (5 μM) Ara-C for 48 h. GAPDH was used as the internal control. (F) Western blotting analysis of BCLAF1 expression levels in AC021683.2 knockdown or control HL60-ADR cells treated with or without MG132 (20 μM) for 8 h in the presence of 5 μM Ara-C. GAPDH was used as the internal control. (G) The ubiquitination of BCLAF1 was analyzed by immunoprecipitation in AC021683.2 -knockdown Flag-BCLAF1 HL60 cells treated with or without (5 μM) Ara-C and western blot with indicated antibodies. Cell lysates were subjected to immunoblot with FLAG tag antibody. (H) AC021683.2 depleted or control HL60-ADR cells were treated with or without (5 μM) Ara-C for 48 h, and immunoprecipitations were performed with indicated antibodies. ns, no significance.

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Knockdown, Ubiquitin Proteomics, Quantitative RT-PCR, Silver Staining, Control, Mass Spectrometry, Western Blot, Expressing, Immunoprecipitation, FLAG-tag

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: BCLAF1 suppresses chemosensitivity of HL60-ADR cells to Ara-C (A) BCLAF1 expression in AML and normal group from GEPIA database. (B) Association between survival of AML patients and BCLAF1 expression levels from the TCGA database. p values were assessed using Gehan-Breslow-Wilcoxon test. (C) RT-qPCR analysis of mRNA levels of BCLAF1 in control and BCLAF1- overexpressing HL60 cells. (D) Western blotting analysis of BCLAF1 protein levels in control and BCLAF1-overexpressing HL60 cells. (E) RT-qPCR analysis of mRNA levels of BCLAF1 in control and BCLAF1 inhibited HL60-ADR cells. (F) Western blotting analysis of BCLAF1 protein levels in control and BCLAF1 knockdown HL60-ADR cells. (G) BCLAF1 overexpressed HL60 cells or control cells were treated with different concentrations of Ara-C for 48 h, the cell viability was detected by CCK-8 assay and the IC50 was calculated. (H) BCLAF1 depleted HL60-ADR cells or control cells were treated with different concentrations of Ara-C for 48 h, the cell viability was detected by CCK-8 assay and the IC50 was calculated. (I) BCLAF1 overexpressed HL60 cells or control cells were treated with or without Ara-C (5 μM) for 48 h, the cell apoptosis was detected by flow cytometry. (J) BCLAF1 knockdown HL60-ADR cells or control cells were treated with or without (5 μM) Ara-C for 48 h, the cell apoptosis was measured by flow cytometry. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-tailed Student’s t tests (C, E) and two-way ANOVA (G-J).

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Knockdown, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: AC021683.2 depletion enhances the chemosensitivity of HL60-ADR cells to Ara-C partially by depending on BCLAF1 (A) The cell viability was detected by CCK8 assay in AC021683.2 knockdown HL60-ADR cells with BCLAF1 overexpression stimulated with different concentrations of Ara-C for 48 h. (B) The cell viability was detected by CCK8 assay in BCLAF1 overexpressed HL60 cells with AC021683.2 knockdown stimulated with different concentrations of Ara-C for 48 h. (C) The indicated cell lines (control, AC021683.2 shRNA, AC021683.2 shRNA with BCLAF1 overexpression) were stimulated with (5 μM) Ara-C for 48 h, the cell apoptosis was measured by flow cytometry. (D) The indicated cell lines (control, BCLAF1 overexpression, BCLAF1 overexpression with AC021683.2 shRNA) were treated with (5 μM) Ara-C for 48 h, the cell apoptosis was measured by flow cytometry. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-way ANOVA (A–D).

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: CCK-8 Assay, Knockdown, Over Expression, Control, shRNA, Flow Cytometry

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: AC021683.2 or BCLAF1 is positively correlated with RAD50 in AML (A) The KEGG pathway enrichment analysis for the targets of the identified AC021683.2 from ENCORI database. (B) RT-qPCR analysis of mRNA levels of BRCA1 , ATM , RAD51 , RAD54 , and RAD50 in control and BCLAF1 suppressed HL60-ADR cells in the presence of 5 μM Ara-C. (C) RT-qPCR analysis of mRNA levels of BRCA1 , ATM , RAD51 , RAD54 , and RAD50 in control and AC021683.2 inhibited HL60-ADR cells in the presence of 5 μM Ara-C. (D) Co-expression analysis of RAD50 and BCLAF1 was performed in AML samples based on the ENCORI database. (E) Co-expression analysis of RAD50 and BCLAF1 was performed in AML samples based on the GEPIA database. (F) Co-expression analysis of AC021683.2 and RAD50 in AML samples was performed based on the ENCORI database. (G) Co-expression analysis of AC021683.2 and RAD50 in AML samples was performed based on the GEPIA database. (H) Pearson correlations between AC021683.2 and RAD50 expression levels in AML patient samples. (I) Western blotting analysis of BCLAF1 and RAD50 protein levels in control and BCLAF1 knockdown HL60-ADR cells in the presence of 5 μM Ara-C, respectively. (J) Western blotting analysis of BCLAF1 and RAD50 protein levels in AC02683.2 suppressed HL60-ADR cells and control cells under stimulation with 5 μM Ara-C. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-way ANOVA (B–C). ns, no significance.

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Quantitative RT-PCR, Control, Expressing, Western Blot, Knockdown

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: RAD50 mediates the role of AC021683.2 and BCLAF1 in Ara-C-induced HL60-ADR cells apoptosis (A) RT-qPCR analysis of mRNA levels of RAD50 in control and RAD50 suppressed HL60-ADR cells. (B) Western blotting analysis of RAD50 protein levels in HL60-ADR cells with RAD50 knockdown and control cells. (C) RAD50 depleted HL60-ADR cells or control cells were treated with different concentrations of Ara-C for 48 h, respectively, the cell viability was detected by CCK-8 assay and the IC50 was calculated. (D) RAD50 knockdown HL60-ADR cells or control cells were treated with (5 μM) Ara-C for 48 h, respectively, the cell apoptosis was measured by Flow cytometry. (E) Western blotting analysis of RAD50 protein levels and BCLAF1-Flag in BCLAF1 overexpressed HL60 cells with RAD50 knockdown and control cells. (F) The cell viability was detected by CCK8 assay in BCLAF1 overexpressed HL60 cells with RAD50 knockdown stimulated with different concentrations of Ara-C for 48 h. (G) The indicated cell lines (Control, BCLAF1 overexpression, BCLAF1 overexpression with RAD50 shRNA) were treated with (5 μM) Ara-C for 48 h, respectively, the cell apoptosis was measured by flow cytometry. (H) Western blotting analysis of RAD50 protein levels in AC021683.2 knocked down HL60-ADR cells with RAD50 overexpression and control cells. (I) The cell viability was detected by CCK8 assay in AC021683.2 knocked down HL60-ADR cells with RAD50 overexpression stimulated with different concentrations of Ara-C for 48 h. (J) The indicated cell lines (Control, AC021683.2 shRNA, AC021683.2 shRNA with RAD50 overexpression) were stimulated with (5 μM) Ara-C for 48 h, respectively, the cell apoptosis was measured by Flow cytometry. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-tailed Student’s t tests (A) and two-way ANOVA (C-D, F-G, and I-J). ns, no significance.

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Quantitative RT-PCR, Control, Western Blot, Knockdown, CCK-8 Assay, Flow Cytometry, Over Expression, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Data from mass spectrometry of HEK-293T cells demonstrating Bclaf1 on Flag-FERM-PTK2 immunoprecipitate. B. Interaction networks of PTK2 and Bclaf1 on HEK293T obtained from IntAct ( https://www.ebi.ac.uk/intact/home ), including association and physical association. C-D . Bclaf1 immunoblots of PTK2 immunoprecipitated from control H9c2 extracts confirming the PTK2-Bclaf1 interaction, and vice versa. PTK2 (C) and Bclaf1 (D) served as loading control. E. Super-resolution images of H9c2 cells on basal conditions, stained for PTK2, Bclaf1, actin, and nucleus. Scale bar = 10 μm. Nucleus zoomed image scale bar = 5 μm. F-G. Z-stack slices of nuclear planes showing the association of PTK2 with Bclaf1 (arrows). Scale bar = 1 μm. Zoomed images scale bar = 0.1μm. H. Proximity Ligation Assay (PLA) demonstrating the interaction between PTK2 and Bclaf1 in untreated cells. Scale bar: 5 μm. Green: PTK2; Red: Bclaf1; Blue: nucleus; Grey: actin; magenta: PLA.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Control, Staining, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Triple-label SR-SIM image showing PTK2 and Bclaf1 concentrated on biomolecular condensates on the nucleus of H9c2 cardiomyocytes treated with dox. Scale bar = 10 µm. 1. Fluorescence line profile plot showing PTK2 colocalizing with ubiquitin outside the Bclaf1 condensate. Scale bar = 0.5 µm. 2. Fluorescence line profile plot showing low ubiquitin level inside the Bclaf1 condensate, where PTK2 is enriched. Scale bar = 0.5 µm B. Pearson correlation coefficient between Bclaf1 and PTK2, ubiquitin and PTK2, and ubiquitin and Bclaf1. C. PTK2 immunoblots of extracts from control (CT) and dox-treated (DOX) H9c2 cells and bar graph showing a decrease in PTK2 levels after dox treatment. D. RT-qPCR assay shows no difference in PTK2 mRNA levels in cardiomyocytes on basal conditions or after dox treatment. GAPDH was used as endogenous control. E. Western-blot of PTK2 and ubiquitin comparing soluble (S) and insoluble (P) fractions of H9c2 cardiomyocytes. F. Super-resolution images of PTK2, Bclaf1, and ubiquitin staining after 36 hours of recovery from dox treatment. Scale bar = 5 µm. 1. Fluorescence line profile plot showing low PTK2 ubiquitination inside Bclaf1 condensate. Scale bar = 0.5 µm. 2. Fluorescence line profile plot showing PTK2 ubiquitinated and agglomerated outside Bclaf1 condensate. G. Graphical representation of PTK2-Bclaf1 undergoing LLPT to avoid PTK2 ubiquitination and aggregation. H. Western-blot showing ubiquitin accumulation on cardiomyocytes treated with dox and co-treated with dox and MG132. I. SR-SIM image showing the accumulation of ubiquitinated PTK2 on the nucleus of cardiomyocytes co-treated with dox and MG132. Scale bar of nuclear image = 5 µm. (1) Orthogonal Z projection showing yz planes and xz planes of a PTK2 agglomerate, which colocalize with ubiquitin. Scale bar = 0.5 µm. J. Cell number count after dox combined with MG132 treatment, as indicated. Green: PTK2; Red: Bclaf1; Grey: Ubiquitin. Asterisks demonstrates statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Fluorescence, Ubiquitin Proteomics, Western Blot, Control, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A-B. Immunofluorescence images of H9c2 cardiomyocytes showing PTK2 and Bclaf1 reorganization in dot-like structures after dox treatment, respectively. C. Immunofluorescence images of pY397-PTK2 (pPTK2) showing nuclear PTK2 activation after dox treatment. D. Bar graphs show the nuclear fluorescence intensity of PTK2, Bclaf1, and pPTK2 on basal conditions and after dox treatment. Asterisks means statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001. E-F. pY397-PTK2, PTK2, GAPDH, and Histone H1 (H1) specific immunoblots of cytoplasmic and nuclear extract of H9c2 control or treated with dox. Dox treatment: 1 µM; 12h. Green: PTK2 and pPTK2; Grey: Actin; Red: Bclaf1; Blue: nucleus. Scale bar = 5 µm. Scale bar of zoomed images = 3 µm.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Immunofluorescence, Activation Assay, Fluorescence, Western Blot, Control

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A-B . Bclaf1 immunoblots of PTK2 immunoprecipitate from dox-treated H9c2 cardiomyocytes extracts, and vice versa. PTK2 (A) and Bclaf1 (B) served as input loading control. C. Immunofluorescence images of dox-treated cardiomyocytes showing the rearrangement of Bclaf1 into nuclear condensates containing PTK2. Below, Z-stack from nuclear planes, demonstrating the association of PTK2 and Bclaf1 in different planes. Scale bar of upper panels = 5 µm, 1 µm, 2 µm, and 0.5 µm, respectively. Scale bar of Z-stack images = 0.5 µm. D. PTK2-Bclaf1 condensate image showing strong colocalization in contrast with a no-complex region. Arrowhead shows the 3 PTK2 bright peaks. Scale bar = 0.5 µm. E. Orthogonal Z projection showing yz planes, xz planes and 3D reconstruction of the Bclaf1 biomolecular condensate containing PTK2 shown in “d”. F. Fluorescence line profile plot of the yellow horizontal line in (E) showing PTK2 inside the Bclaf1 condensate. Green: PTK2; Red: Bclaf1; Blue: nucleus. Dox treatment: 1 µM; 12h. G. Proximity Ligation Assay showing increased interaction between PTK2 and Bclaf1 after dox treatment. Scale bar = 5 µm.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Western Blot, Control, Immunofluorescence, Fluorescence, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. The structure of Bclaf1, an intrinsically disordered protein (IDP), with an inner core of 4 alpha helices and flexible long loops was obtained using AlphaFold3. PAE matrix indicates low error values only on the diagonal, reflecting the Bclaf1 unfolding nature. B. PTK2 structure, with three main domains and a long intrinsically disordered region (IDR) linking the Kinase and FAT domains obtained by AlphaFold3 platform is shown. PAE matrix shows the predicted error in Angstroms. C. Predicted PTK2-Bclaf1 modeled using AlphaFold3 docking tool and PAE matrix, indicating low error values for the PTK2 domains and a higher error value for the IDR. Interfaces of PTK2-Bclaf1 interaction with intermediate error values are shown. D. LLPS propensity scores for Bclaf1, PTK2, IDR PTK2-N-terminal, Q686 – S923 IDR PTK2 sequence, and the PTK2-Bclaf1 complex obtained using FuzDrop and PSPredictor. E. Residue-based droplet promoting probability of PTK2 and Bclaf1 obtained by FuzDrop.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. FRAP fitted graphs of condensate and out of condensate nuclear regions of H9c2 cells transfected with EGFP-Bclaf1 and EGFP-PTK2, in individual experiments. Bclaf1 and PTK2 present higher diffusion on condensate regions in relation to non-condensate areas. Images show the fluorescence and differential interference contrast (DIC) images of EGFP-Bclaf1 and EGFP-PTK2 expressing cells. Scale bar = 5 μm. B. Mobile fraction and constant of the fluorescence recovery rate for Bclaf1 and PTK2. C. Lifetime images of H9c2 cells expressing GFP-Bclaf1 or GFP-PTK2 fluorescent proteins. D. Bar graph of EGFP-Bclaf1 and EGFP-PTK2 fluorescence lifetime in condensates and no condensate areas. Asterisks means statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Transfection, Diffusion-based Assay, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Super-resolution images of H9c2 cardiomyocytes showing the colocalization of Hsp70 (gray) with PTK2 (green) and Bclaf1 (red) on the condensate region. Scale bar = 10 µm. Scale bar of zoomed images = 0.5 µm. B. FRAP graph showing the fluorescence recovery after photobleaching of EGFP-Bclaf1 and EGFP-PTK2 after Hsp70 inhibition (iHsp70). Asterisks demonstrates statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001. C. Time-course experiments using CellROX show an increasing oxidative stress during dox treatment. Scale bar = 5 µm. D. Bar Graph shows the percentage of cells with Bclaf1 clusters from control (CT) to 12h of dox treatment, as indicated (n=80 for CT, n=59 for 1h, n=94 for 3h, n=76 for 6h, and n=91 for 12h). E. Bar graph shows the MTT absorbance at 540 nm. F. Western Blot images of p53, p53 phosphorylated at Ser 15 (p-p53-S15), and cleavage caspase-3. α-Tubulin served as loading control. Bar Graphs are shown in Supplementary Figure 4.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Fluorescence, Inhibition, Control, Western Blot

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. MS/MS spectrum showing the PTK2 identified peptide (m/z 561.3040) with the chemical modification (K-GG) characteristic of ubiquitination. B. Linear representation of rat and human PTK2 demonstrating their domains and the PTK2 ubiquitination site on rat, lysine 926, and in human, lysine 923. C. Cartoon representation of PTK2 showing the K926 ubiquitination site on the FAT domain. D. Hydrostatic representation of PTK2-Bclaf1 interaction complex, showing K926 site being sterically occluded by Bclaf1. The FERM domain is represented in blue, the Kinase in red, and the FAT domain in yellow.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Tandem Mass Spectroscopy, Modification, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Western blot image and bar graph confirming the knockdown of Bclaf1 (KD-Bclaf1) in relation to control (CT) H9c2 cardiomyocytes. B. Western blotting images show the increased ubiquitination after Bclaf1 knockdown in relation to control cardiomyocytes, both treated with dox. C. SR-SIM image and bar graph show the ubiquitin accumulation on the nucleus of KD-Bclaf1 cardiomyocytes treated with dox. Scale bar = 5 µm. D. SR-SIM image shows the effect of Bclaf1 knockdown on disrupting condensates assembly. Scale bar = 5 µm. E. SR-SIM image and line profile plot showing Bclaf1 knockdown increases PTK2 agglomeration and ubiquitination on the nucleus. Scale bar = 5 µm. Scale Bar of zoomed image = 0.5 µm. 1. Fluorescence line profile plot showing low PTK2 ubiquitination in PTK2 condensate. 2. Fluorescence line profile plot showing PTK2 ubiquitinated outside the PTK2 condensate. F. Western Blot and bar graphs show PTK2 levels reduction on KD-Bclaf1 cells treated with dox, while PTK2 mRNA expression levels increased. G. Western blot of soluble extracts from control and KD-Bclaf1 H9c2 cells treated with dox. Bar graphs show a reduction of PTK2-Full length (PTK2-FL) band and an increase in PTK2 cleaved bands (55 KDa and 34 kDa). H. Western blot and bar graph analysis of insoluble extract from control and KD-Bclaf1 H9c2 cells treated with dox. The analysis showed no difference between CT and KD-Bclaf1 cells on the amount of PTK2-FL and PTK2 fragments present on the insoluble fraction. I. Western blot and bar graphs show increased p53 and PUMA levels on KD-Bclaf1 H9c2 cardiomyocytes treated with dox. On the right, the bar graph shows a reduced number of cells on the KD-Bclaf1 cells in relation to control cells, after dox treatment.

Article Snippet: Then, primary antibodies against PTK2 (Thermo Fisher, ZF002, mouse) and Bclaf1 (Thermo Fisher, PA5-78299, rabbit) were added to a 1:200 concentration and incubated in a humidity chamber at 4°C for 1 hour.

Techniques: Western Blot, Knockdown, Control, Ubiquitin Proteomics, Fluorescence, Expressing

Journal: Translational Oncology

Article Title: Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma

doi: 10.1016/j.tranon.2022.101502

Figure Lengend Snippet: Upregulation of VPS35 levels positively correlates with Bclaf1 levels, poorer prognosis and higher metastasis in HCC. We analyzed data obtained from the CNHPP data portal. Increased protein (A) and mRNA (B) levels of VPS35 and Bclaf1 of patients with HCC. (C) A positive correlation between the protein ( r = 0.5509, P <0.0001) and mRNA ( r = 0.5138, P <0.0001) levels of Bclaf1 and VPS35. (D) Relative expression of Bclaf1 and VPS35 genes in normal tissues and liver hepatocellular carcinoma tissues with different nodal metastasis status, cancer stage, and tumor grade. (E) UALCAN survival analysis of patients with HCC based on VPS35 and Bclaf1 protein level. (F) The protein expression of Bclaf1 and VPS35 in tumor tissue (TT) or adjacent tissue (AT) of patients with HCC was detected by IHC.

Article Snippet: The primary antibodies were as followed: β-actin (66,009–1–1 g, PROTEINTECH), Bclaf1 (26,809–1-AP, Santa Cruz Biotechnology), VPS35 (sc-374,372, Santa Cruz Biotechnology), HSF1 (sc-9144, Santa Cruz Biotechnology), Hsp70 (sc-69,705, Santa Cruz Biotechnology), Hsp90α (8165 s, Cell Signaling Technology), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (25,682–1-AP, PROTEINTECH).

Techniques: Expressing

Journal: Translational Oncology

Article Title: Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma

doi: 10.1016/j.tranon.2022.101502

Figure Lengend Snippet: N-terminal Hsp90 inhibitor STA9090 induced HSF1 activation and increased Bclaf1/VPS35 level in HCC . (A) HepG2 cells were incubated with 0.1μmol/L STA9090 and 0.5 mmol/L NB for 24 h as indicated. Protein levels of Bclaf1, VPS35, Hsp90α, Hsp70, and HSF1 were determined by western blotting and (B) mRNA levels of Bclaf1 and VPS35 were determined by RT-PCR analysis. (C)Confocal immunofluorescence images showed VPS35 (red) and Bclaf1 (green) intensity. (D) Protein levels of Bclaf1 and VPS35 in Control, STA9090, and NB-treated tumors were detected by immunohistochemistry. Image Pro-plus 6.0 software was used to quantify the Mean of IOD and Value represent mean ± SD, n = 6 * P < 0.01, ** P <0.01, *** P < 0.001.

Article Snippet: The primary antibodies were as followed: β-actin (66,009–1–1 g, PROTEINTECH), Bclaf1 (26,809–1-AP, Santa Cruz Biotechnology), VPS35 (sc-374,372, Santa Cruz Biotechnology), HSF1 (sc-9144, Santa Cruz Biotechnology), Hsp70 (sc-69,705, Santa Cruz Biotechnology), Hsp90α (8165 s, Cell Signaling Technology), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (25,682–1-AP, PROTEINTECH).

Techniques: Activation Assay, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Control, Immunohistochemistry, Software

Journal: Translational Oncology

Article Title: Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma

doi: 10.1016/j.tranon.2022.101502

Figure Lengend Snippet: Bclaf1 upregulated VPS35 transcription through bZIP DNA binding domain in HCC cells . (A) The schematic representation of VPS35 promoter with the indicated region (P1) for PCR analysis. HepG2 cell was incubated with STA9090 for 24 h. ChIP-PCR was adopted to measure Bclaf1 binding on VPS35 promoter region. (B, C) HepG2 and Huh7 cells were transiently transfected with siNC, siBclaf1, EV plasmid, and FL plasmid. The Bclaf1 and VPS35 level were measured by western blotting and RT-PCR. (D) HepG2 cells were transfected with siNC and siBclaf1 for 48 h or transfected with EV and FL plasmids for 24 h. ChIP-PCR was adopted to measure Bclaf1 binding on VPS35 promoter region. And HepG2 cells were co-transfected with VPS35 promoter-luciferase reporter plasmids and siNC or siBclaf1 or EV plasmid or FL plasmid. Luciferase reporter gene assay was carried out to detect the interaction between Bclaf1 and VPS35 promoter region. The luciferase activity decreased transiently transfected with siBclaf1 and increased transiently transfected with Bclaf1-FL plasmid. (E) Schematic structure of Bclaf1 domains (upper panel). HepG2 cells were transfected with different Bclaf1-encoding plasmids for 24 h and the transfection efficiency was confirmed by western blot (lower panel). (F, G) HepG2 cells were transiently transfected with EV, FL, ΔMyb, and ΔbZIP plasmids for 24 h. The VPS35 protein and mRNA levels were measured by western blot and RT-PCR. (H) HepG2 cells were transiently transfected with EV and ΔbZIP plasmids for 24 h. ChIP-PCR was adopted to measure Bclaf1 binding on VPS35 promoter region. Image J software v1.8.0 was used to quantify the bands relative intensities and Value represent mean ± SD, n = 3 * P < 0.01, ** P <0.01, *** P < 0.001.

Article Snippet: The primary antibodies were as followed: β-actin (66,009–1–1 g, PROTEINTECH), Bclaf1 (26,809–1-AP, Santa Cruz Biotechnology), VPS35 (sc-374,372, Santa Cruz Biotechnology), HSF1 (sc-9144, Santa Cruz Biotechnology), Hsp70 (sc-69,705, Santa Cruz Biotechnology), Hsp90α (8165 s, Cell Signaling Technology), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (25,682–1-AP, PROTEINTECH).

Techniques: Binding Assay, Incubation, Transfection, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Gene Assay, Activity Assay, Software

Journal: Translational Oncology

Article Title: Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma

doi: 10.1016/j.tranon.2022.101502

Figure Lengend Snippet: N-terminal Hsp90 inhibitor STA9090 promotes HCC metastasis via Bclaf1-VPS35-EVs axis in HCC. (A, B) HepG2 cells treated with NC-siRNA, NC-siRNA plus STA9090, Bclaf1-siRNA, and Bclaf1-siRNA plus STA9090 or treated with NC-siRNA, NC-siRNA plus STA9090, VPS35-siRNA, and VPS35-siRNA plus STA9090 for 48 h. The Bclaf1 and VPS35 levels were detected using western blotting. (C) HepG2 cells were transiently co-transfected with siVPS35 and Bclaf1-FL plasmid for 48 h. The Bclaf1 and VPS35 levels were detected using western blotting. Image J software v1.8.0 was used to quantify the bands relative intensities and Value represent mean ± SD, n = 3 * P < 0.01, ** P <0.01, *** P < 0.001. (D) Transwell invasion assays were performed with HepG2 cells transfected either with siNC, siNC-Bclaf1-FL plasmid, siVPS35, and siVPS35-Bclaf1-FL plasmid. (E) Transwell invasion assays were performed with HepG2 cell incubated with 2 μg of EVs extracted from the culture medium of shNC/shBclaf1 stable lentivirus cell lines either treated with STA9090. Number of cells/field were measured by Image Pro-plus6.0, and Value represent mean ± SD, n = 6 * P < 0.01, ** P <0.01, *** P < 0.001. The scale is 200 μm.

Article Snippet: The primary antibodies were as followed: β-actin (66,009–1–1 g, PROTEINTECH), Bclaf1 (26,809–1-AP, Santa Cruz Biotechnology), VPS35 (sc-374,372, Santa Cruz Biotechnology), HSF1 (sc-9144, Santa Cruz Biotechnology), Hsp70 (sc-69,705, Santa Cruz Biotechnology), Hsp90α (8165 s, Cell Signaling Technology), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (25,682–1-AP, PROTEINTECH).

Techniques: Western Blot, Transfection, Plasmid Preparation, Software, Incubation

Journal: Translational Oncology

Article Title: Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma

doi: 10.1016/j.tranon.2022.101502

Figure Lengend Snippet: Model for the HSF1-Bclaf1-VPS35-EVs connection in HCC . STA9090 treatment activated HSF1 and increased levels of Bclaf1, which promoted the transcription of VPS35 by directly binding to the VPS35 promoter via its bZIP DNA binding domain, consequently leading to the increase of VPS35 protein. Upregulated VPS35 protein promoted the EVs delivery process from Golgi-apparatus, early endosome, late endosome (or Multiple vesicles body), and eventually secreted to extracellular space to exert its function in HCC metastasis.

Article Snippet: The primary antibodies were as followed: β-actin (66,009–1–1 g, PROTEINTECH), Bclaf1 (26,809–1-AP, Santa Cruz Biotechnology), VPS35 (sc-374,372, Santa Cruz Biotechnology), HSF1 (sc-9144, Santa Cruz Biotechnology), Hsp70 (sc-69,705, Santa Cruz Biotechnology), Hsp90α (8165 s, Cell Signaling Technology), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (25,682–1-AP, PROTEINTECH).

Techniques: Binding Assay

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Characterization and expression pattern of Bclaf1 in the developing mouse retina. A, Retinal sections from E14.5 to adulthood mice were stained with a rabbit-polyclonal antibody against Bclaf1. Bclaf1 expression is restricted to the INBL and the GCL from E14.5 to P3 and then to the INL until adulthood. Note that at P3 and later, a few Bclaf1+ cells are also found in the differentiating ONL and could potentially correspond to horizontal cells. The white boxed areas on E14.5 panels are twofold magnifications (top and right corner) to show the faintly labeled cells located in the center of the retina. Scale bars: 60 μm (E14.5), 30 μm (E16.5-adulthood). IPL, Inner plexiform layer; OPL, outer plexiform layer. B, Schematic structure of short and long isoforms of Bclaf1. The black box corresponds to the deleted part in the short isoform of Bclaf1. The epitope recognized by the Bclaf1 polyclonal antibody maps to a region between residues 150 and 200, shared by the two isoforms. C, Expression of the long and short isoforms of Bclaf1 in the retina at E18.5, P3, and P14 detected by PCR using specific primers able to amplify both isoforms (primers a + b) or only the short isoform (primers c + b).

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Expressing, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Expression of Bclaf1 in early-born retinal cells except cone photoreceptors. Sections of central retina close to the optic nerve at E16.5 (A–C), E18.5 (D–G), P0 (H–K), P5 (L–O), P7 (P), and P14 (Q, R) were immunostained for Bclaf1 (red) and various cell-type markers (green). Small panels display the distribution of labeled cells for each specific marker in the retina visualized by DAPI counterstaining (gray). Large panels (merge) show that Bclaf1 is not expressed in postmitotic CyclinD1+ cells (A); note that the Bclaf1+ cell (arrow) visible in the ONBL (A) is cyclinD1-negative unlike virtually all other cells in this layer. At E16.5, nearly all Bclaf1+ cells express Pax6 (B) and all Brn3a+ RGCs express Bclaf1 (C). Conversely, in the ONBL, markers of precursors (Otx2, Crx,) or differentiating (recoverin) photoreceptors do not colocalized at E18.5 with Bclaf1 (D–F). Similarly, RXRγ+ cells located in the upper surface of the ONBL (E18.5), corresponding to cone-photoreceptor precursors, do not express Bclaf1 (G). At P0 and P5, markers of RGCs (Brn3a, I) or amacrine cells, such as AP2 (K, M), Pax6 (H), calretinin (L), and calbindin (J) colocalize with Bclaf1. Horizontal cells, identified at P5 by calbindin (N) or by Lim1 (O) are Bclaf1+. At P7 or P14, late born cells, i.e., rod-photoreceptors (rhodopsin+, P) Müller glial cells (GS+, Q) and bipolar cells (PKCα+, R) do not express Bclaf1. Scale bars, 30 μm.

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Expressing, Labeling, Marker

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Expression of Bclaf1 in the adult retina. Retinal sections from adult mice were visualized by DAPI counterstaining (blue) and double-stained for Bclaf1 (red) and for the indicated marker (green). Confocal microscopic acquisitions show that Bclaf1 is expressed in Pax6+ cells, corresponding to RGCs (Brn3a), ACs (calretinin, calbindin), and HCs (Lim1, calbindin). Bclaf1 never colocalized with Rhodopsin, a rod-photoreceptor marker, PKCα, a bipolar cell marker, or Sox9, a marker of Müller glial cells. Scale bar, 30 μm.

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Expressing, Staining, Marker

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Early but not late lineage is affected in Bclaf1-deficient mice embryos. Immunostaining for Pax6 (red), cyclinD1, PCNA, Otx2, Crx, or recoverin (green) on retinal sections from WT and Bclaf1-deficient mice at E16.5 (A) or E18.5 (B, C). Sections were counterstained with DAPI (gray). The density of Pax6+ cells, corresponding to early-born cell populations, is reduced in absence of Bclaf1 at E16.5 and E18.5. At the same stages, no difference is observed in the density of mitotic cells (progenitors) identified with CyclinD1 or PCNA between Bclaf1−/− and WT retina. The density of precursors of late born retinal cells (identified with Otx2, Crx, or recoverin) is not modified at E18.5 in Bclaf1−/− retina. Scale bar, 30 μm.

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Immunostaining, Modification

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Bclaf1-deficient retinas display defects in the development of early-born cells. A–F, Retinal sections from E14.5 to E18.5 Bclaf1−/− mice or WT littermate were stained for Brn3a (A), calbindin (B), or RXRγ (C). The density (number of cells per mm) of Brn3a+ RGCs is lower in Bclaf1−/− compared with WT retina at E14.5 (minus 55.9%), E16.5 (minus 35.5%), but not at E18.5 (D). The density of calbindin+ ACs in the innermost part of the INBL and calbindin+ HCs located in the ONBL is reduced at E18.5 in Bclaf1−/− retina (minus 26% and 36.7%, respectively) (E). The density of cone-photoreceptor precursors (RXRγ+ cells) is increased by 93% in Bclaf1−/− compared with WT retina at E18.5 (F). RXRγ+ cells were quantified in the ONBL only (dotted line delimited area) to exclude nonphotoreceptor RXRγ+ cells. G, Sections from retinal explants (E18.5 + 7 DIV) of Bclaf1−/− mice or WT littermate were stained for Lim1, calretinin, calbindin, or cone arrestin (green) and counterstained for DAPI (gray). H, The density of Lim1+ (HCs), calretinin+ (ACs), and calbindin+ cells (HCs and ACs) at E18.5 + 7 DIV was reduced in Bclaf1−/− retinal explants compared with WT, whereas the density of CAR+ cells (cone photoreceptors) was higher. Error bars indicate SEM and an asterisk a significant statistical difference (*p < 0.05, **p < 0.01; n = 4–5). Scale bars, 30 μm.

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Bclaf1 silencing in early rat RPCs reproduces the retinal phenotype of Bclaf1-deficient mice. A, Rat retinal sections from the indicated developmental stages were immunolabeled with a Bclaf1 rabbit-polyclonal antibody (red) and weakly counterstained with nuclear DAPI (gray). Bclaf1 expression is restricted to the INBL and both the INL and GCL at different developmental stages. B–E, Retinal explants from E16 rats were electroporated with either pU6-shBclaf1-eGFP or pU6shLuciferase-eGFP (control) vectors. After 5 DIV, dissociated cells (B) or frozen sections (C–E) from retinal explants were double-immunostained with an anti-GFP antibody and antibodies against the indicated cell type. Silencing Bclaf1 led to a decrease in positive cells for Pax6, AP2, calretinin, Lim1, and calbindin among GFP+ cells (B). No differences in cell death were observed after silencing Bclaf1 compared with control (B). Sections stained for GFP and either Pax6 (C), calretinin (D), or Lim1 (E) showed a decrease in double-labeled cells in shBclaf1-electroporated explants. Arrows indicate colabeled cells. Values represent the mean ± SEM (*p < 0.05, **p < 0.01) from at least three separate retina counts. Scale bar, 50 μm.

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Immunolabeling, Expressing, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Molecular mechanisms involved in Bclaf1 effects. A, Schematic structure of Bclaf1 mutants. The black box corresponds to the sequence that is lacking in the short isoform. The gray boxes represent the putative bZip and Myb DNA binding domains. B, Expression of the two mutants of Bclaf1, cloned into pCIG-eGFP vector and transfected in DF1 cells. Western-blotting analysis confirmed the production of the two mutant forms DC210 and DN384 at the predicted sizes of 25 and 54 kDa, respectively. Polyclonal and monoclonal antibodies recognized epitopes corresponding to region from residues 150–200 (full form and DC210; left) and 318–439 (full form and DN384; right) respectively. C, Analysis of subcellular localization of the two Bclaf1 mutants in DF1 cells by immunofluorescence. Note that the DN384 mutant is located exclusively in the cytoplasm in contrast to the full-length form of Bclaf1 and the DC210 mutant, which both display nuclear localization. D, Retinal explants from P0 pups were electroporated with either pCIG-Bclaf1-eGFP, pCIG-DC210-eGFP, pCIG-DN384-eGFP or pCIG-eGFP (control) vectors. After 7 DIV, dissociated cells from retinal explants were double-immunostained with an anti-GFP antibody and antibodies against Pax6 or rhodospin. Only overexpression of DC210 mutant leads to the same effects that full-length form of Bclaf1. E, BHK21 cells were transiently transfected with a reporting vector driving expression of CAT under the control of the glucagon promoter (which is Pax6-dependent), the Pax6-expressing vector, and one of the vectors expressing the distinct mutants of Bclaf1. Bclaf1 alone is unable to transactivate the glucagon promoter but a synergistic effect on the transactivation of the glucagon promoter is observed between Pax6 and Bclaf1 when they are coexpressed, except in the case of the DN384 mutant. The results expressed as mean ± SEM from at least three different experiments were normalized to β-galactosidase activity derived from a cotransfected CMV-LacZ expression plasmid. F, q-PCR on mRNA extracts from E16.5 retina showed that Ascl1a and Ngn2 relative expression was reduced in Bclaf1−/− compared with WT embryos. No modification of expression was observed for the Atoh7 relative expression. Data are expressed as mean ± SEM from three different experiments (*p < 0.05, ***p < 0.001).

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Sequencing, Binding Assay, Expressing, Clone Assay, Plasmid Preparation, Transfection, Western Blot, Mutagenesis, Immunofluorescence, Over Expression, Activity Assay, Derivative Assay, Modification

Journal: The Journal of Neuroscience

Article Title: Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

doi: 10.1523/JNEUROSCI.3227-13.2014

Figure Lengend Snippet: Ectopic expression of Bclaf1 in postnatal retinal explants is not sufficient to bias the cell fate of late progenitors and induces the cell death of differentiating rod photoreceptors. Retinal explants from P0 pups were electroporated with either pCIG-Bclaf1-eGFP or pCIG-eGFP (control) vectors. A, After 7 DIV, dissociated cells from retinal explants were coimmunostained for GFP with Pax6, Calretinin or Calbindin. The graph shows the ratio of specific marker+ cells from the GFP+ cell population. Forced expression of Bclaf1 induced an increase by 45% of Pax6+ cell ratio within GFP+ cell population, compared with control. The ratio of calretinin+ or calbindin+ cells displays no significant difference between the two experimental groups. B, q-PCR on mRNA extracts from FACS sorted GFP-positive cells 36 h after electroporation with pCIG-Bclaf1-eGFP or pCIG-eGFP. Bclaf1 overexpression is confirmed by the sixfold increase of its relative expression compared with control conditions. By contrast, no significant modification of expression was found for transcription factors required for AC or HC specification such FoxN4, Ptf1, and Prox1. C, Forced expression of Bclaf1 induced a 55.7% decrease of rhodopsin+/GFP+ cell ratio in P0 + 7 DIV retinal explants compared with control. D, E, After 7 DIV, cells undergoing apoptosis were TUNEL-labeled in retinal sections (E). Quantitative analysis of GFP+ apoptotic cells in the INL and ONL per section (D) showed a significant increase in TUNEL+ cells in pCIG-Bclaf1-eGFP electroporated explants compared with control explants. Values (A–D) represent the mean ± SEM (*p < 0.05, **p < 0.01) from at least three separate retina counts. Scale bars, 50 μm.

Article Snippet: The following primary antibodies were used: mouse anti-AP2 (DSHB), mouse anti-Brn3a (Millipore), mouse and rabbit anti-Bclaf1 (BD Biosciences and Bethyl), rabbit anti-Calbindin (Swant), mouse anti-Calretinin (Millipore), rabbit anti-Cone arrestin (CAR; Millipore), mouse anti-CyclinD1 (Santa Cruz Biotechnology), mouse and rabbit anti-GFP (Roche and Clinisciences), mouse anti-glutamine synthetase (GS; Millipore), mouse anti-Lim1 (DSHB); rabbit anti-Otx2 (Millipore), rabbit anti-Pax6 (Millipore), rabbit anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) rabbit anti-Protein Kinase Cα (PKCα, Santa Cruz Biotechnology), mouse anti-rhodopsin (R4D2, gift from Dr Molday, University of British Columbia, Vancouver, Canada), rabbit anti-recoverin (Millipore), rabbit anti-Retinoid acid receptor gamma (RXRγ; Santa Cruz Biotechnology), and rabbit anti-Sox9 (Millipore).

Techniques: Expressing, Marker, Electroporation, Over Expression, Modification, TUNEL Assay, Labeling

Journal: Frontiers in Pharmacology

Article Title: Phosphoproteomic and proteomic profiling in post-infarction chronic heart failure

doi: 10.3389/fphar.2023.1181622

Figure Lengend Snippet: Intersection of differentially expressed phosphorylated proteins and Thanatos Apoptosis Database (A) Venn diagram of data in Thanatos Apoptosis Database and differentially expressed phosphorylated proteins (DPPs). (B) Venn diagram of identified apoptosis-related DPPs and hub DPPs. (C,D) Bclaf1 Ser658 expression in left ventricular from post-infarction chronic heart failure rats and sham operation (SO) rats.

Article Snippet: The membranes were blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated for 8 h at 4°C with rabbit Bclaf1 Ser658 antibody (Abmart), protein kinase catalytic subunit alpha-1 gene (PRKAA1) antibody (Proteintech), protein kinase A catalytic α subunit (PRKACA) antibody (Proteintech), protein kinase D1 (PKD1) antibody (Immunoway), p21-activated kinase 1 (PAK1) antibody (Immunoway), protein kinase C delta (PRKCD) antibody (Proteintech), and glyceraldehyde 3–phosphate dehydrogenase (GAPDH) antibody (Abmart).

Techniques: Expressing