Journal: JCI Insight

Article Title: Elevation of master autophagy regulator Tfeb in osteoblast lineage cells increases bone mass and strength

doi: 10.1172/jci.insight.191688

Figure Lengend Snippet: ( A – E ) Skeletal phenotype analysis of 4.5-month-old male Tfeb CRa mice and their littermate Cre controls (Osx1-Cre only [red dots], Osx1-Cre CRa [blue dots], Osx1-Cre sgRNA Tfeb [gray dots]). ( A – C ) Dynamic histomorphometry was performed on femurs. n = 5–6 mice per group. ( A ) Quantification of mineralizing surface per bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate per bone surface (BFR/BS) at the periosteal surface. ( B ) Representative histological cross section showing calcein labeling at the femoral diaphysis (GFP filter). Scale bars represent 400 μm. ( C ) Trabecular MAR (Tb. MAR). ( D and E ) Sp7 and Col1a1 ( D ) and Acp5 and Ctsk ( E ) mRNA levels were measured in L5 by qRT-PCR and normalized to mouse Actb . n = 18 mice per group (please see Methods). ( F and G ) Bone marrow cells were isolated from femurs and tibias of 5-month-old female mice and differentiated into osteoblasts using osteogenic media. ( F ) Alizarin red stain of mineral apposition. ( G ) BrdU analysis of proliferating cells. n = 4 wells per group. ( H ) Circulating sclerostin levels of 5- and 12-month-old male (square) and female (circle) Tfeb CRa and control mice were measured using ELISA. n = 5–7 mice per group. ( I ) Sclerostin and actin levels were measured in cortical bone (humeri shafts) of Tfeb CRa and control mice via Western blot. Immunoblot of 5 mice per group is shown. Quantification was done with n = 10–11 mice per group. The bar graph indicates sclerostin levels normalized to actin. Bars indicate mean ± SD. Indicated P values were calculated by unpaired t test for equal or unequal variance ( A -Ps.MAR).

Article Snippet: Circulating sclerostin levels were measured using Mouse/Rat SOST/Sclerostin Quantikine ELISA Kit (R&D Systems, MSST00).

Techniques: Labeling, Quantitative RT-PCR, Isolation, Staining, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: JCI Insight

Article Title: Elevation of master autophagy regulator Tfeb in osteoblast lineage cells increases bone mass and strength

doi: 10.1172/jci.insight.191688

Figure Lengend Snippet: ( A – C ) Serial spine ( A ), femur ( B ), and global (whole body) ( C ) DXA BMD measurements were collected from female Tfeb CRa mice ( n = 6) and their littermate Cre controls (Osx1-Cre only, Osx1-Cre sgRNA Tfeb mice, n = 8) between 3 and 12 months of age. Error bars indicate SD. * P < 0.05 as calculated by unpaired t test at each time point. Indicated P values were calculated by repeated measures model (detailed in the Methods section) comparing the difference in genotypes at 3 versus 9 months or 9 versus 12 months. ( D and E ) μCT analysis of Ct.Th ( D ) and trabecular BV/TV ( E ) performed on lumbar vertebrae 4 (spine) and femurs from 12-month-old female Tfeb CRa mice ( n = 6) and littermate controls (Osx1-Cre only [red dots], Osx1-Cre sgRNA Tfeb [gray dots], n = 8). ( F ) ELISA measurements of P1NP and TRAcP 5b ( Tfeb CRa n = 6, controls n = 7). ( G – J ) Histomorphometric analysis of vertebral trabecular bone of Tfeb CRa mice ( n = 5) and their Cre controls ( n = 8). ( G ) MS/BS, BFR/BS. ( H ) Osteoblast surface per bone perimeter (Ob.S/B.Pm), osteoblast number per bone perimeter (N.Ob/B.Pm). ( I ) Osteoclast surface per bone perimeter (Oc.S/B.Pm), osteoclast number per bone perimeter (N.Oc/B.Pm). ( J ) TRAcP 5b– and toluidine blue–stained or unstained (labeled with calcein and alizarin red) sections imaged with 40× original magnification. Scale bars represent 50 μm. ( K and L ) Bglap and Sp7 ( K ) and Acp5 and Ctsk ( L ) mRNA levels measured in lumbar vertebrae 5 (spine) of Tfeb CRa mice ( n = 7) and controls ( n = 6) using qRT-PCR and normalized to mouse Actb . Bars indicate mean ± SD. Indicated P values were calculated by unpaired t test for equal or unequal variance ( E -femur and spine, K - Sp7 ) or rank sum test (if data are not normally distributed, D -femur, H –N.Ob/B.Pm).

Article Snippet: Circulating sclerostin levels were measured using Mouse/Rat SOST/Sclerostin Quantikine ELISA Kit (R&D Systems, MSST00).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Labeling, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

doi: 10.1074/jbc.m610929200

Figure Lengend Snippet: FIGURE 2. ProBMP1SSQQ binds BMP4 and BMP2 in a highly specific man- ner. A, an immunoblot is shown of samples containing () or lacking () 5 nM proBMP1SSQQ, BMP1, and/or BMP4; which were immunoprecipitated with anti-BMP1 antibodies (26). Blots were cut and the separate pieces were incu- bated with anti-BMP1 or anti-BMP4 antibodies. B, proBMP1SSQQ was incu- bated with a fixed amount of BMP4 (5 nM) alone, or in the presence of increas- ing amounts of BMP2 (molar ratios of BMP4:BMP2 of 1:1, 1:2, 1:5, and 1:10) followed by immunoprecipitation with anti-BMP1 antibody (26) and immu- noblotting with anti-BMP4 or anti-BMP2 monoclonal antibodies. As a control for pull-down efficiency, the anti-BMP4 blot was stripped and re-probed with anti-BMP1 antibodies to detect immunoprecipitated proBMP1SSQQ. C, puri- fied BMP1 prodomain, produced in a baculovirus system, was visualized by stainingwithCoomassieBrilliantBlueR-250(lane2).Numberstotheleftofgel correspond to the approximate sizes in kDa of molecular mass markers (lane 1). D, Western blot is used for characterization of anti-BMP prodomain anti- bodies, which at a 1:20,000 dilution detect 6 ng of BMP1-Fc fusion protein (lane 1), but do not detect 6 ng of Fc domain (lane 2).

Article Snippet: Recombinant BMP2, BMP4, BMP5, EGF, BMPR-1A/Fc, and TGF- 1 were all purchased from R&D systems.

Techniques: Western Blot, Immunoprecipitation, Bioprocessing, Control, Produced

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

doi: 10.1074/jbc.m610929200

Figure Lengend Snippet: FIGURE 3. The BMP1 prodomain directly binds BMP2 and BMP4 with high specificity and affinity. A, 5 nM BMP4 and His-tagged BMP1 prodomain were incubated separately or together, followed by precipitation with nickel- chargedaffinityresin(leftpanel)orwithanti-BMP1prodomainantibody(right panel) and immunoblot analysis with anti-prodomain or anti-BMP4 antibod- ies.B,5nMBMP2andHis-taggedBMP1prodomainwereincubatedseparately or together, followed by precipitation with anti-BMP1 prodomain antibody and protein A-Sepharose, and immunoblot analysis with anti-BMP1 prodo- main or with anti-BMP2 antibodies. C, 5 nM BMP4 and His-tagged BMP1 prodomain were coincubated, as in A, but in the presence of 10-fold excesses ofBMP2,TGF-1,EGF,orBMP5;followedbyprecipitationwithnickel-charged affinity resin and immunoblot analyses. D, 5 nM BMP4 and soluble BMP recep- tor IA (BMPR-1A, ALK-3), fused to an Fc domain, were coincubated in the presence of 1:1, 2:1, or 5:1 molar ratios of either BMP1 prodomain or Chordin, and the samples then precipitated with protein A-Sepharose and analyzed by immunoblots employing anti-BMP4 or anti-BMPR-1A antibodies.

Article Snippet: Recombinant BMP2, BMP4, BMP5, EGF, BMPR-1A/Fc, and TGF- 1 were all purchased from R&D systems.

Techniques: Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

doi: 10.1074/jbc.m610929200

Figure Lengend Snippet: FIGURE 4. The BMP1 prodomain binds BMP2 with a KD of 10.9 nM. Coomassie Brilliant Blue R-250-stained SDS-PAGE gels are shown for molecular mass standards (lane 1) and purified BMP1 prodomain-Fc fusion protein (lane 2) (A) or molecular mass standards (lane 1) and purified BMP1 prodomain minus the Fc domain (lane 2) (B). Numbers to the left of gels correspond to the approximate sizes in kDa of molecular mass markers. BMP1 prodomain (C) and murine Chordin (D) bind to BMP2 with KDs of 10.9 4.7 nM and 6.7 1.0 nM, respectively.

Article Snippet: Recombinant BMP2, BMP4, BMP5, EGF, BMPR-1A/Fc, and TGF- 1 were all purchased from R&D systems.

Techniques: Staining, SDS Page, Purification

Journal: DNA repair

Article Title: Quantitative Analysis of ATM Phosphorylation in Lymphocytes

doi: 10.1016/j.dnarep.2019.06.002

Figure Lengend Snippet: H2AX activation in human PBMCs. (A) Comparison of H2AX and ATM activation in 41 volunteers following 2 Gy IR (Spearman correlation coefficient ρ = −0.09, p-value=0.58). (B) H2AX activation in females versus males (p = 0.042).

Article Snippet: Antibodies: phospho-S1981-ATM (clone EP1890Y, cat#:ab81292, Abeam); pan-ATM (clone 2C1, cat#GTX70103, GeneTex); phosphoserine-139 histone H2AX (clone 20E3, cat#9718, Cell Signaling); histone H2AX (clone 322105, cat#MAB3406, R&D Systems); IRDye-800CW (cat#926-32211, LI-COR Biotechnology-US); IRDye-680RD (cat#926-68070, LI-COR).

Techniques: Activation Assay

Journal: Biology

Article Title: Effect of Recombinant NGF Encapsulated in Chitosan on Rabbit Sperm Traits and Main Metabolic Pathways

doi: 10.3390/biology14080974

Figure Lengend Snippet: Effect of chitosan and NGF + chitosan on JNK phosphorylation in rabbit sperm traits. ( A ) Immunoblot of Phospho-JNK and total JNK after 30 min of treatment with chitosan alone or with rrβNGFch. ( B ). Densitometric analysis of Phospho-JNK/JNK/β-Tubulin after 30 min of treatments. ( C ) Immunoblot of Phospho-JNK and total JNK after 2 h of treatment with chitosan alone or with rrβNGFch. ( D ) Densitometric analysis of Phospho JNK/JNK/β-Tubulin after 2 h. The corresponding full-length Western blots are shown in .

Article Snippet: Membranes were incubated for 5 min in EveryBlot Blocking Buffer (Bio-Rad Laboratories S.r.l.) and then overnight with primary antibodies that included Phospho-AKT (Ser473) (D9E) XP ® Rabbit mAb (#4060, 1:1000, Cell Signaling Technology, CST), anti- AKT Antibody (#9272, 1:1000, Cell Signaling Technology, CST), Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Antibody (#9101, 1:1000, Cell Signaling Technology, CST), p44/42 MAPK (ERK1/2)(#9102, 1:1000, Cell Signaling Technology, CST anti-Phospho-JNK (T183/Y185) Antibody (#AF1205, 1:1000, R&D Systems), anti- JNK Pan Specific Antibody (#AF1387, 1:1000, R&D Systems), and β-Tubulin Antibody (#2146, 1:1000, Cell Signaling Technology, (CST), Danvers, MA, USA) diluted in EveryBlot Blocking Buffer (Bio-Rad Laboratories S.r.l.) or BSA (Sigma-Aldrich).

Techniques: Phospho-proteomics, 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