Journal: bioRxiv

Article Title: βA3/A1-crystallin is an epigenetic regulator of histone deacetylase 3 (HDAC3) in the retinal pigmented epithelial (RPE) cells

doi: 10.1101/2024.08.06.606634

Figure Lengend Snippet: Cryba1 deletion diminishes HDAC3 activity in mouse RPE cells. (A) Schematic diagram of global HDAC activity assay. Nuclear lysate was isolated followed by measuring HDAC activity fluorometrically in a 96-well dark half area opaque microtiter plate. BOC-(Ac)-Lyc-AMC was used as a substrate. (B) The cell lysate was isolated from Cryba1 fl/fl and Cryba1 cKO mouse RPE cells, followed by a class I HDAC activity assay. Deletion of Cryba1 in the cKO RPE diminished the enzymatic activity of Class I HDAC. The result was represented as fold change compared with blank value and representative of three individual experiments. (n=3, ***p<0.001). (C) Schematic depicting immunoprecipitation of selective HDAC followed by activity assay. HDAC1 (D) , HDAC2 (E) , HDAC3 (F), and HDAC8 (G) were immunoprecipitated from Cryba1 fl/fl and Cryba1 cKO mouse RPE cells followed by evaluation of activity assay for the respective HDACs. HDAC3 activity was decreased in Cryba1 cKO compared to Cryba1 fl/fl (F) . The activity of HDAC1/2 and 8 in Cryba1 cKO was comparable to Cryba1 fl/fl . IgG was used as a negative control. Data has been presented as fold change compared with IgG. Results are representative of three individual experiments. (n=3, ****p<0.0001, NS= not significant). (H) Heatmap of enzymatic activity of all class I HDACs (Histone deacetylase) and histone acetylase p300. (I) Immunoblot analysis of HDAC1, HDAC2, HDAC3, and HDAC8 from the whole cell lysate isolated from Cryba1 fl/fl and Cryba1 cKO mouse RPE cells demonstrate comparable amounts of protein levels in the RPE cells from both genotypes. Actin (cytosolic) and lamin (nuclear) were used as loading controls. The result was representative of three individual experiments. (n=3). (J) HDAC3 was immuno-precipitated from Cryba1 fl/fl , βA1 KD and βA3 KO RPE cells, followed by a HDAC3 activity assay. HDAC3 activity significantly decreased in both βA1 KD and βA3 KO cells, while βA1 KD showed a greater reduction in HDAC3 activity than βA3 KO cells. IgG was used as negative control. Data has been presented as fold change compared with IgG value. (n=3, ****p<0.0001 Vs βA1 KD, **p<0.01 Vs βA3 KO). (K) Immunoblot analysis of HDAC3 from the total RPE lysate isolated from Cryba1 fl/fl , βA1 KD and βA3 KO. The result was representative of three individual experiments. (n=3). (L) NCor1, which is an HDAC3 complex protein, was immuno-precipitated from Cryba1 fl/fl and Cryba1 cKO RPE cell lysates followed by HDAC3 activity. The data demonstrates a marked loss of HDAC3’s enzymatic activity in Cryba1 cKO compared to Cryba1 fl/fl . IgG was used as a negative control. Data has been presented as fold change compared with IgG value. The result was representative of three individual experiments. (n=3, ****p<0.0001). (M) SMRT, which is another HDAC3 complex protein, was immuno-precipitated from Cryba1 fl/fl and Cryba1 cKO RPE cell lysates followed by HDAC3 activity. The data demonstrates loss of enzymatic activity of HDAC3 in Cryba1 cKO compared to Cryba1 fl/fl . IgG was used as a negative control. Data has been presented as fold change compared with IgG value. The result was representative of three individual experiments. (n=3, ****p<0.0001). (N) Immunoblot analysis of NCor1 and SMRT from the total lysates isolated from Cryba1 fl/fl and Cryba1 cKO RPE cells demonstrating comparable protein levels in both lysates, while actin was used as a loading control. The result was representative of three individual experiments. (n=3)

Article Snippet: About 20 μg total protein from the lysate was incubated against anti-p300 CBP HAT antibody (Cell Signaling Technology, Cat #: 86377) respectively at 4°C for 1 hr, followed by capturing the antibody with EZview A/G beads (Millipore-Sigma, Cat #: E3403).

Techniques: Activity Assay, HDAC Activity Assay, Isolation, Immunoprecipitation, Negative Control, Histone Deacetylase Assay, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: BDNF reverses aging-related microglial activation

doi: 10.1186/s12974-020-01887-1

Figure Lengend Snippet: BDNF inhibited microglial activation via upregulation of Erk-CREB signaling. a , b Representative immunofluorescent images show the distribution of p-CREB and p-p65 in the BV-2 cells. Scale bar, 10 μm. c Interaction of CBP with p-CREB and p-p65, assessed by immunoprecipitation of CBP followed by immunoblot for p-CREB and p-p65. d , e Levels of p-Erk ( n = 4) and p-CREB ( n = 4) in the BV2 cells. f Levels of TNF ( n = 4) and IL-6 ( n = 4) in conditioned media. * p < 0.05, ** p < 0.01, *** p < 0.001 versus LPS(-)BDNF(-)PD98059(-) group; # p < 0.05, , ### p < 0.001 versus LPS(+)BDNF(-)PD98059(-) group; $ P < 0.05, $$ P < 0.01 versus LPS(+)BDNF(+)PD98059(-) group. g – i Levels of p-Akt ( n = 4), p-GSK3-β ( n = 4) and MKP-1 ( n = 4) in the BV2 cells. * p < 0.05, ** p < 0.01, *** p < 0.001 versus LPS(-)BDNF(-) group; # p < 0.05 versus LPS(+)BDNF(-) group. Data are presented as mean ± S.D

Article Snippet: Pre-cleared lysates were then subjected to immunoprecipitation using anti-CREB-binding protein (CBP) antibodies (Cat. #: 7389S, Cell Signaling Technology) and protein A/G PLUS-Dynabeads (Invitrogen) for 16 h at 4 °C.

Techniques: Activation Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A) Lgals3 mRNA expression relative to Rpl13a measured by RTqPCR in the cerebellum during development. E17: embryonic day 17. P0, P7, P14, and P21: postnatal day 0, 7, 14, and 21. (B) Lgals3 knockout (KO) mice present no defects in cerebellar morphology. Parasagittal cerebellar slices of wild-type (WT) and Lgals3 KO adult mice were stained using the nuclear marker Hoechst.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Expressing, Knock-Out, Staining, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A and B) Immunostaining for the Purkinje cell marker CABP (green) and the presynaptic marker VGLUT2 (red) was performed on sections from Lgals3 WT and KO mice at P15 (A) and at P65 (B). (C and D) Immunostaining for the Purkinje cell marker CABP (green) and the presynaptic marker VGLUT1 (red) specific for mature parallel fiber synapses was performed on sections from Lgals3 WT and KO mice at P15 (C) and at P65 (D). Scale bar: 25μm. Data are representative of three independent experiments.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A) Immunohistochemistry for LGALS3 show the specificity of the LGALS3 antibody. WT: wild-type mice; KO: knockout mice. Positive cells in the molecular layer are marked with a white arrowhead, scale bar 50μm. (B) Immunohistochemistry for the Purkinje cell marker CABP (green) and LGALS3 (red) on parasagittal cerebellar sections from P15 and P22 wild-type mice. Scale bar=100μm. Data are representative of three independent experiments. Legends: 4V, 4 th ventricle; chp, choroid plexus; pcg, pontine central gray and mv, medial vestibular nucleus.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Immunohistochemistry, Knock-Out, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A) LGALS3 expression in oligodendrocytes in the cerebellar white matter. Co-immunostaining of parasagittal cerebellar slices using anti-LGALS3 (red), oligodendrocyte marker anti-OLIG-2 (green) and the nuclear marker Hoechst (blue). (B) LGALS3 expression in astrocytes in the cerebellar white matter. Co-immunostaining of parasagittal cerebellar slices using anti-LGALS3 (red), astrocyte marker anti-GFAP (green) and the nuclear marker Hoechst (blue). (C) LGALS3 expression in some microglial cells of the molecular layer. Immunostaining anti-LGALS3 (red) in parasagittal sections of mice CX 3 CR1 eGFP/eGFP , microglial CX 3 CR1-GFP (green) and the nuclear marker Hoechst (blue). Scale bar 50μm and 20μm for the magnification. Data are representative of three independent experiments. Legends: 4V, 4 th ventricle; GL, granular layer; ML, molecular layer and WM, white matter.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Expressing, Immunostaining, Marker

Journal: iScience

Article Title: Prenatal low-dose methylmercury exposure causes premature neuronal differentiation and autism-like behaviors in a rodent model

doi: 10.1016/j.isci.2023.106093

Figure Lengend Snippet:

Article Snippet: For PLA, the primary antibodies were rabbit anti- p -CREB (S133) (cat#9198S, Cell Signaling 1:500) and mouse anti-CBP (cat#sc-7300, Santa Cruz Biotechnology, 1:200).

Techniques: Recombinant, Proximity Ligation Assay, In Situ, Software, Microscopy

Journal: Biomolecules

Article Title: Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes.

doi: 10.3390/biom13121732

Figure Lengend Snippet: Figure 1. Western blot analysis of lysosome purity. The lysosomes were isolated from the control and imipramine-treated mexAMs using density gradient ultracentrifugation and compared to the cell lysate (cells). Protein of equal weight (10 µg) was loaded into each lane. The bands represent the organelle-specific antibodies: lysosomes (LIMP2), ER (Calreticulin), Golgi, (Golgin97), and late endosomes (Rab7). Two PVDF membranes were imaged simultaneously under the same conditions to produce a single image (Figure S3).

Article Snippet: The primary antibodies were used to detect specific organelle markers as follows: lysosome Biomolecules 2023, 13, 1732 4 of 15 (LIMP2/SCARB2 Rabbit mAb, cat # 27960, Cell Signaling, Danvers, MA, USA), ER (Calreticulin Rabbit Ab, cat # 2891, Cell Signaling), Golgi (Golgin-97 Rabbit mAb, cat # 13192, Cell Signaling), and late endosome (Rab7 Rabbit mAb, cat # 9367, Cell Signaling).

Techniques: Western Blot, Isolation, Control