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polyclonal antibodies for hem1  (Novus Biologicals)


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    Novus Biologicals polyclonal antibodies for hem1
    Deletion of <t>Hem1</t> increases trabecular bone mass in mice. Micro-CT imaging and quantification of femoral bones from male Hem1 knockout mice and wildtype littermates at 5.5 weeks of age. A , representative images of whole femur ( left ) and length measurement ( right ). B , representative images of cortical bone at midshaft (scale bar represents 100 μm). C , cortical thickness and cortical perimeter at midshaft. D , representative images of trabecular bone (scale bar represents 1 mm) and ( E ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. F , histological sections of distal femurs stained with Masson's trichrome. G , standard histomorphometric parameters quantifying trabecular architecture (BV/TV, trabecular number, thickness, and spacing) in histologic sections of 5.5-week-old male mice. Lines and error bars represent mean ± SD; n = 4 to 5 animals/group. p Values were determined with Student’s t test. Hem1, hematopoietic protein 1.
    Polyclonal Antibodies For Hem1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal antibodies for hem1/product/Novus Biologicals
    Average 92 stars, based on 2 article reviews
    polyclonal antibodies for hem1 - by Bioz Stars, 2026-02
    92/100 stars

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    1) Product Images from "Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice"

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.102841

    Deletion of Hem1 increases trabecular bone mass in mice. Micro-CT imaging and quantification of femoral bones from male Hem1 knockout mice and wildtype littermates at 5.5 weeks of age. A , representative images of whole femur ( left ) and length measurement ( right ). B , representative images of cortical bone at midshaft (scale bar represents 100 μm). C , cortical thickness and cortical perimeter at midshaft. D , representative images of trabecular bone (scale bar represents 1 mm) and ( E ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. F , histological sections of distal femurs stained with Masson's trichrome. G , standard histomorphometric parameters quantifying trabecular architecture (BV/TV, trabecular number, thickness, and spacing) in histologic sections of 5.5-week-old male mice. Lines and error bars represent mean ± SD; n = 4 to 5 animals/group. p Values were determined with Student’s t test. Hem1, hematopoietic protein 1.
    Figure Legend Snippet: Deletion of Hem1 increases trabecular bone mass in mice. Micro-CT imaging and quantification of femoral bones from male Hem1 knockout mice and wildtype littermates at 5.5 weeks of age. A , representative images of whole femur ( left ) and length measurement ( right ). B , representative images of cortical bone at midshaft (scale bar represents 100 μm). C , cortical thickness and cortical perimeter at midshaft. D , representative images of trabecular bone (scale bar represents 1 mm) and ( E ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. F , histological sections of distal femurs stained with Masson's trichrome. G , standard histomorphometric parameters quantifying trabecular architecture (BV/TV, trabecular number, thickness, and spacing) in histologic sections of 5.5-week-old male mice. Lines and error bars represent mean ± SD; n = 4 to 5 animals/group. p Values were determined with Student’s t test. Hem1, hematopoietic protein 1.

    Techniques Used: Micro-CT, Imaging, Knock-Out, Staining

    Deletion of Hem1 in mice decreases bone resorption. A , representative photomicrographs of osteoclasts ( left ), number of osteoclasts ( middle ; N.Oc/B.Pm), and nuclei number per osteoclast ( right ; N.Nuclei/Oc) per trabecular bone surface of nondecalcified femur sections stained for TRAP activity ( red arrows ) from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 400 μm). Dotted red lines indicate multinucleated TRAP-positive osteoclasts. B , ELISA analysis of serum concentration of a collagen degradation product (CTx) in 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group). C , representative photomicrographs of osteoblasts ( left ; black arrows ) and number of osteoblasts ( right ; N.Ob/B.Pm) per trabecular bone surface of nondecalcified femur sections stained for Masson's trichrome from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 100 μm). D , ELISA analysis of serum concentration of osteocalcin (n = 3–4 animals/group). E – G , photomicrographs show representative colonies ( top ). CFU-F stained for alkaline phosphatase after 10 days, CFU-AD stained with Oil Red O after 7 days, and CFU-OB stained with von Kossa after 25 days to detect mineral ( bottom ) (triplicate cultures) (scale bar represents 1 cm). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. CFU-AD, CFU adipocyte; CFU-F, CFU fibroblast; CFU-OB, CFU-osteoblast; Hem1, hematopoietic protein 1; TRAP, tartrate-resistant acid phosphatase.
    Figure Legend Snippet: Deletion of Hem1 in mice decreases bone resorption. A , representative photomicrographs of osteoclasts ( left ), number of osteoclasts ( middle ; N.Oc/B.Pm), and nuclei number per osteoclast ( right ; N.Nuclei/Oc) per trabecular bone surface of nondecalcified femur sections stained for TRAP activity ( red arrows ) from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 400 μm). Dotted red lines indicate multinucleated TRAP-positive osteoclasts. B , ELISA analysis of serum concentration of a collagen degradation product (CTx) in 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group). C , representative photomicrographs of osteoblasts ( left ; black arrows ) and number of osteoblasts ( right ; N.Ob/B.Pm) per trabecular bone surface of nondecalcified femur sections stained for Masson's trichrome from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 100 μm). D , ELISA analysis of serum concentration of osteocalcin (n = 3–4 animals/group). E – G , photomicrographs show representative colonies ( top ). CFU-F stained for alkaline phosphatase after 10 days, CFU-AD stained with Oil Red O after 7 days, and CFU-OB stained with von Kossa after 25 days to detect mineral ( bottom ) (triplicate cultures) (scale bar represents 1 cm). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. CFU-AD, CFU adipocyte; CFU-F, CFU fibroblast; CFU-OB, CFU-osteoblast; Hem1, hematopoietic protein 1; TRAP, tartrate-resistant acid phosphatase.

    Techniques Used: Staining, Activity Assay, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Deletion of Hem1 decreases osteoclast fusion. A and B , bone marrow macrophages were isolated from 6-month-old C57BL/6 wildtype mice and were cultured with M-CSF (30 ng ml −1 , bone marrow macrophages) or with M-CSF and RANKL (30 ng ml −1 ) for 2 days (pOC) or 5 days (mOC). Levels of Hem1 ( A ) mRNA (quantitative PCR [qPCR] assay) and ( B ) protein (Western blot) during osteoclastogenesis. C – H , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF (30 ng ml −1 ) and RANKL (30 ng ml −1 ) for ( C ) 24 h, ( D , F , and G) 5 days, ( H ) 3 days, or ( E and H ) indicated periods. C , Hem1 mRNA levels (qPCR assay) during osteoclastogenesis. D , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages (scale bar represents 500 μm). E , mRNA levels (qPCR assay) of osteoclast markers during osteoclastogenesis. F , representative pictures of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). The resorbed areas appear white , and the unresorbed mineralized surface appears black . G , representative pictures of DAPI ( blue ; nuclei) and phalloidin ( red ; actin rings) staining of osteoclast cultures (scale bar represents 500 μm). H , mRNA levels (qPCR assay) of osteoclast fusion markers during osteoclastogenesis. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined with ( A ) one-way ANOVA or ( C – H ) Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.
    Figure Legend Snippet: Deletion of Hem1 decreases osteoclast fusion. A and B , bone marrow macrophages were isolated from 6-month-old C57BL/6 wildtype mice and were cultured with M-CSF (30 ng ml −1 , bone marrow macrophages) or with M-CSF and RANKL (30 ng ml −1 ) for 2 days (pOC) or 5 days (mOC). Levels of Hem1 ( A ) mRNA (quantitative PCR [qPCR] assay) and ( B ) protein (Western blot) during osteoclastogenesis. C – H , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF (30 ng ml −1 ) and RANKL (30 ng ml −1 ) for ( C ) 24 h, ( D , F , and G) 5 days, ( H ) 3 days, or ( E and H ) indicated periods. C , Hem1 mRNA levels (qPCR assay) during osteoclastogenesis. D , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages (scale bar represents 500 μm). E , mRNA levels (qPCR assay) of osteoclast markers during osteoclastogenesis. F , representative pictures of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). The resorbed areas appear white , and the unresorbed mineralized surface appears black . G , representative pictures of DAPI ( blue ; nuclei) and phalloidin ( red ; actin rings) staining of osteoclast cultures (scale bar represents 500 μm). H , mRNA levels (qPCR assay) of osteoclast fusion markers during osteoclastogenesis. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined with ( A ) one-way ANOVA or ( C – H ) Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Techniques Used: Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Generated, Staining

    Rescue of cell fusion in Hem1-deficient osteoclasts by retroviral transduction of Hem1. Bone marrow macrophages lacking Hem1 were transduced with retroviral vectors expressing either empty vector (MSCV) or Hem1 (MSCV-Hem1) and were cultured with M-CSF and RANKL for ( A ) 24 h or ( B – D ) 5 days. A , protein levels (Western blot) of Hem1 in bone marrow macrophage cell cultures. B , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei, generated from bone marrow macrophages (scale bar represents 500 μm). C , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). D , mRNA levels (quantitative PCR assay) of osteoclast markers in mOCs. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined using Student’s t test. Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; MSCV, murine stem cell virus; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.
    Figure Legend Snippet: Rescue of cell fusion in Hem1-deficient osteoclasts by retroviral transduction of Hem1. Bone marrow macrophages lacking Hem1 were transduced with retroviral vectors expressing either empty vector (MSCV) or Hem1 (MSCV-Hem1) and were cultured with M-CSF and RANKL for ( A ) 24 h or ( B – D ) 5 days. A , protein levels (Western blot) of Hem1 in bone marrow macrophage cell cultures. B , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei, generated from bone marrow macrophages (scale bar represents 500 μm). C , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). D , mRNA levels (quantitative PCR assay) of osteoclast markers in mOCs. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined using Student’s t test. Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; MSCV, murine stem cell virus; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Techniques Used: Retroviral, Transduction, Expressing, Plasmid Preparation, Cell Culture, Western Blot, Generated, Staining, Real-time Polymerase Chain Reaction, Virus

    Deletion of Hem1 attenuates respiration by suppressing c-Abl signaling. A – F , bone marrow macrophages from indicated genotypes were cultured with M-CSF and RANKL for 3 days. Different fractions of mitochondrial and nonmitochondrial respiration per 10,000 cells, in osteoclasts, were measured by Seahorse XF96 (n = 11–12 wells/group). G – I , bone marrow macrophages lacking Hem1 were cultured with M-CSF and RANKL for ( G ) 3 days or ( H and I ) indicated time points. G , ATP levels in osteoclasts (RLU, relative luminescence units; n = 4/group). H and I , protein levels (Western blot) in bone marrow macrophage cultures. To clarify Hem1 deletion in osteoclasts, the same images of Hem1 band are used. These are the same set of samples from the bone marrow macrophage cultures. J , osteoclasts developed in cultures of bone marrow macrophages from 6-month-old male C57BL/6 mice in the presence or the absence of imatinib (5 μM). Representative pictures ( left ), number ( middle ), and total area ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (triplicate cultures). K and L , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF and RANKL for ( K ) 5 or ( L ) 3 days in the presence or the absence of DPH (1 μM). K , number of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages. L , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis. Lines and error bars represent mean ± SD. p Values were determined with ( A – G ) Student’s t test, ( J ) one-way ANOVA, or ( K and L ) two-way ANOVA. DPH, 5-(1, 3-diaryl-1H-pyrazol-4-yl); Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.
    Figure Legend Snippet: Deletion of Hem1 attenuates respiration by suppressing c-Abl signaling. A – F , bone marrow macrophages from indicated genotypes were cultured with M-CSF and RANKL for 3 days. Different fractions of mitochondrial and nonmitochondrial respiration per 10,000 cells, in osteoclasts, were measured by Seahorse XF96 (n = 11–12 wells/group). G – I , bone marrow macrophages lacking Hem1 were cultured with M-CSF and RANKL for ( G ) 3 days or ( H and I ) indicated time points. G , ATP levels in osteoclasts (RLU, relative luminescence units; n = 4/group). H and I , protein levels (Western blot) in bone marrow macrophage cultures. To clarify Hem1 deletion in osteoclasts, the same images of Hem1 band are used. These are the same set of samples from the bone marrow macrophage cultures. J , osteoclasts developed in cultures of bone marrow macrophages from 6-month-old male C57BL/6 mice in the presence or the absence of imatinib (5 μM). Representative pictures ( left ), number ( middle ), and total area ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (triplicate cultures). K and L , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF and RANKL for ( K ) 5 or ( L ) 3 days in the presence or the absence of DPH (1 μM). K , number of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages. L , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis. Lines and error bars represent mean ± SD. p Values were determined with ( A – G ) Student’s t test, ( J ) one-way ANOVA, or ( K and L ) two-way ANOVA. DPH, 5-(1, 3-diaryl-1H-pyrazol-4-yl); Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Techniques Used: Cell Culture, Western Blot, Isolation, Knock-Out, Generated, Real-time Polymerase Chain Reaction

    Rescue of bone phenotype in Hem1-null mice by transplantation with wildtype hematopoietic cells. Hematopoietic stem cells from GFP + Lin − CD45 + wildtype mice efficiently repopulated the hematopoietic system of nonablated Hem1-deficient mice after bone marrow cell transplantation. Femurs and bone marrow were analyzed 10 weeks after transplantation (n = 5). A , schematic model of bone marrow transplantation. B , representative images of whole body ( left ) and longitudinal weight measurement ( right ). C , representative images of whole femur ( left ) and length measurement in 13-week-old male recipient Hem1 knockout mice and wildtype littermates ( right ). D , representative images of cortical bone at midshaft (scale bar represents 100 μm). E , cortical thickness and cortical perimeters at the midshaft. F , representative images of trabecular bone (scale bar represents 1 mm) and ( G ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. H , serum concentration of a collagen degradation product (CTx) by ELISA. I , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (triplicate cultures). J – M , bone marrow macrophages were isolated from 13-week-old recipient Hem1 knockout mice and wildtype littermates and cultured with M-CSF and RANKL for ( G , I , and J ) 5 days or ( K and M ) 3 days. J , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (n = 6). K , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis (triplicate cultures). L , representative pictures of DAPI ( blue , nuclei) and phalloidin ( red , actin rings) staining in osteoclast cultures (scale bar represents 500 μm). M , mRNA levels (quantitative PCR assay) of fusion markers during osteoclastogenesis (triplicate cultures). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.
    Figure Legend Snippet: Rescue of bone phenotype in Hem1-null mice by transplantation with wildtype hematopoietic cells. Hematopoietic stem cells from GFP + Lin − CD45 + wildtype mice efficiently repopulated the hematopoietic system of nonablated Hem1-deficient mice after bone marrow cell transplantation. Femurs and bone marrow were analyzed 10 weeks after transplantation (n = 5). A , schematic model of bone marrow transplantation. B , representative images of whole body ( left ) and longitudinal weight measurement ( right ). C , representative images of whole femur ( left ) and length measurement in 13-week-old male recipient Hem1 knockout mice and wildtype littermates ( right ). D , representative images of cortical bone at midshaft (scale bar represents 100 μm). E , cortical thickness and cortical perimeters at the midshaft. F , representative images of trabecular bone (scale bar represents 1 mm) and ( G ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. H , serum concentration of a collagen degradation product (CTx) by ELISA. I , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (triplicate cultures). J – M , bone marrow macrophages were isolated from 13-week-old recipient Hem1 knockout mice and wildtype littermates and cultured with M-CSF and RANKL for ( G , I , and J ) 5 days or ( K and M ) 3 days. J , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (n = 6). K , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis (triplicate cultures). L , representative pictures of DAPI ( blue , nuclei) and phalloidin ( red , actin rings) staining in osteoclast cultures (scale bar represents 500 μm). M , mRNA levels (quantitative PCR assay) of fusion markers during osteoclastogenesis (triplicate cultures). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Techniques Used: Transplantation Assay, Knock-Out, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Cell Culture, Real-time Polymerase Chain Reaction



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    Deletion of Hem1 increases trabecular bone mass in mice. Micro-CT imaging and quantification of femoral bones from male Hem1 knockout mice and wildtype littermates at 5.5 weeks of age. A , representative images of whole femur ( left ) and length measurement ( right ). B , representative images of cortical bone at midshaft (scale bar represents 100 μm). C , cortical thickness and cortical perimeter at midshaft. D , representative images of trabecular bone (scale bar represents 1 mm) and ( E ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. F , histological sections of distal femurs stained with Masson's trichrome. G , standard histomorphometric parameters quantifying trabecular architecture (BV/TV, trabecular number, thickness, and spacing) in histologic sections of 5.5-week-old male mice. Lines and error bars represent mean ± SD; n = 4 to 5 animals/group. p Values were determined with Student’s t test. Hem1, hematopoietic protein 1.

    Journal: The Journal of Biological Chemistry

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    doi: 10.1016/j.jbc.2022.102841

    Figure Lengend Snippet: Deletion of Hem1 increases trabecular bone mass in mice. Micro-CT imaging and quantification of femoral bones from male Hem1 knockout mice and wildtype littermates at 5.5 weeks of age. A , representative images of whole femur ( left ) and length measurement ( right ). B , representative images of cortical bone at midshaft (scale bar represents 100 μm). C , cortical thickness and cortical perimeter at midshaft. D , representative images of trabecular bone (scale bar represents 1 mm) and ( E ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. F , histological sections of distal femurs stained with Masson's trichrome. G , standard histomorphometric parameters quantifying trabecular architecture (BV/TV, trabecular number, thickness, and spacing) in histologic sections of 5.5-week-old male mice. Lines and error bars represent mean ± SD; n = 4 to 5 animals/group. p Values were determined with Student’s t test. Hem1, hematopoietic protein 1.

    Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

    Techniques: Micro-CT, Imaging, Knock-Out, Staining

    Deletion of Hem1 in mice decreases bone resorption. A , representative photomicrographs of osteoclasts ( left ), number of osteoclasts ( middle ; N.Oc/B.Pm), and nuclei number per osteoclast ( right ; N.Nuclei/Oc) per trabecular bone surface of nondecalcified femur sections stained for TRAP activity ( red arrows ) from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 400 μm). Dotted red lines indicate multinucleated TRAP-positive osteoclasts. B , ELISA analysis of serum concentration of a collagen degradation product (CTx) in 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group). C , representative photomicrographs of osteoblasts ( left ; black arrows ) and number of osteoblasts ( right ; N.Ob/B.Pm) per trabecular bone surface of nondecalcified femur sections stained for Masson's trichrome from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 100 μm). D , ELISA analysis of serum concentration of osteocalcin (n = 3–4 animals/group). E – G , photomicrographs show representative colonies ( top ). CFU-F stained for alkaline phosphatase after 10 days, CFU-AD stained with Oil Red O after 7 days, and CFU-OB stained with von Kossa after 25 days to detect mineral ( bottom ) (triplicate cultures) (scale bar represents 1 cm). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. CFU-AD, CFU adipocyte; CFU-F, CFU fibroblast; CFU-OB, CFU-osteoblast; Hem1, hematopoietic protein 1; TRAP, tartrate-resistant acid phosphatase.

    Journal: The Journal of Biological Chemistry

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    doi: 10.1016/j.jbc.2022.102841

    Figure Lengend Snippet: Deletion of Hem1 in mice decreases bone resorption. A , representative photomicrographs of osteoclasts ( left ), number of osteoclasts ( middle ; N.Oc/B.Pm), and nuclei number per osteoclast ( right ; N.Nuclei/Oc) per trabecular bone surface of nondecalcified femur sections stained for TRAP activity ( red arrows ) from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 400 μm). Dotted red lines indicate multinucleated TRAP-positive osteoclasts. B , ELISA analysis of serum concentration of a collagen degradation product (CTx) in 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group). C , representative photomicrographs of osteoblasts ( left ; black arrows ) and number of osteoblasts ( right ; N.Ob/B.Pm) per trabecular bone surface of nondecalcified femur sections stained for Masson's trichrome from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 100 μm). D , ELISA analysis of serum concentration of osteocalcin (n = 3–4 animals/group). E – G , photomicrographs show representative colonies ( top ). CFU-F stained for alkaline phosphatase after 10 days, CFU-AD stained with Oil Red O after 7 days, and CFU-OB stained with von Kossa after 25 days to detect mineral ( bottom ) (triplicate cultures) (scale bar represents 1 cm). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. CFU-AD, CFU adipocyte; CFU-F, CFU fibroblast; CFU-OB, CFU-osteoblast; Hem1, hematopoietic protein 1; TRAP, tartrate-resistant acid phosphatase.

    Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

    Techniques: Staining, Activity Assay, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Deletion of Hem1 decreases osteoclast fusion. A and B , bone marrow macrophages were isolated from 6-month-old C57BL/6 wildtype mice and were cultured with M-CSF (30 ng ml −1 , bone marrow macrophages) or with M-CSF and RANKL (30 ng ml −1 ) for 2 days (pOC) or 5 days (mOC). Levels of Hem1 ( A ) mRNA (quantitative PCR [qPCR] assay) and ( B ) protein (Western blot) during osteoclastogenesis. C – H , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF (30 ng ml −1 ) and RANKL (30 ng ml −1 ) for ( C ) 24 h, ( D , F , and G) 5 days, ( H ) 3 days, or ( E and H ) indicated periods. C , Hem1 mRNA levels (qPCR assay) during osteoclastogenesis. D , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages (scale bar represents 500 μm). E , mRNA levels (qPCR assay) of osteoclast markers during osteoclastogenesis. F , representative pictures of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). The resorbed areas appear white , and the unresorbed mineralized surface appears black . G , representative pictures of DAPI ( blue ; nuclei) and phalloidin ( red ; actin rings) staining of osteoclast cultures (scale bar represents 500 μm). H , mRNA levels (qPCR assay) of osteoclast fusion markers during osteoclastogenesis. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined with ( A ) one-way ANOVA or ( C – H ) Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Journal: The Journal of Biological Chemistry

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    doi: 10.1016/j.jbc.2022.102841

    Figure Lengend Snippet: Deletion of Hem1 decreases osteoclast fusion. A and B , bone marrow macrophages were isolated from 6-month-old C57BL/6 wildtype mice and were cultured with M-CSF (30 ng ml −1 , bone marrow macrophages) or with M-CSF and RANKL (30 ng ml −1 ) for 2 days (pOC) or 5 days (mOC). Levels of Hem1 ( A ) mRNA (quantitative PCR [qPCR] assay) and ( B ) protein (Western blot) during osteoclastogenesis. C – H , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF (30 ng ml −1 ) and RANKL (30 ng ml −1 ) for ( C ) 24 h, ( D , F , and G) 5 days, ( H ) 3 days, or ( E and H ) indicated periods. C , Hem1 mRNA levels (qPCR assay) during osteoclastogenesis. D , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages (scale bar represents 500 μm). E , mRNA levels (qPCR assay) of osteoclast markers during osteoclastogenesis. F , representative pictures of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). The resorbed areas appear white , and the unresorbed mineralized surface appears black . G , representative pictures of DAPI ( blue ; nuclei) and phalloidin ( red ; actin rings) staining of osteoclast cultures (scale bar represents 500 μm). H , mRNA levels (qPCR assay) of osteoclast fusion markers during osteoclastogenesis. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined with ( A ) one-way ANOVA or ( C – H ) Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

    Techniques: Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Generated, Staining

    Rescue of cell fusion in Hem1-deficient osteoclasts by retroviral transduction of Hem1. Bone marrow macrophages lacking Hem1 were transduced with retroviral vectors expressing either empty vector (MSCV) or Hem1 (MSCV-Hem1) and were cultured with M-CSF and RANKL for ( A ) 24 h or ( B – D ) 5 days. A , protein levels (Western blot) of Hem1 in bone marrow macrophage cell cultures. B , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei, generated from bone marrow macrophages (scale bar represents 500 μm). C , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). D , mRNA levels (quantitative PCR assay) of osteoclast markers in mOCs. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined using Student’s t test. Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; MSCV, murine stem cell virus; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Journal: The Journal of Biological Chemistry

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    doi: 10.1016/j.jbc.2022.102841

    Figure Lengend Snippet: Rescue of cell fusion in Hem1-deficient osteoclasts by retroviral transduction of Hem1. Bone marrow macrophages lacking Hem1 were transduced with retroviral vectors expressing either empty vector (MSCV) or Hem1 (MSCV-Hem1) and were cultured with M-CSF and RANKL for ( A ) 24 h or ( B – D ) 5 days. A , protein levels (Western blot) of Hem1 in bone marrow macrophage cell cultures. B , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei, generated from bone marrow macrophages (scale bar represents 500 μm). C , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). D , mRNA levels (quantitative PCR assay) of osteoclast markers in mOCs. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined using Student’s t test. Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; MSCV, murine stem cell virus; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

    Techniques: Retroviral, Transduction, Expressing, Plasmid Preparation, Cell Culture, Western Blot, Generated, Staining, Real-time Polymerase Chain Reaction, Virus

    Deletion of Hem1 attenuates respiration by suppressing c-Abl signaling. A – F , bone marrow macrophages from indicated genotypes were cultured with M-CSF and RANKL for 3 days. Different fractions of mitochondrial and nonmitochondrial respiration per 10,000 cells, in osteoclasts, were measured by Seahorse XF96 (n = 11–12 wells/group). G – I , bone marrow macrophages lacking Hem1 were cultured with M-CSF and RANKL for ( G ) 3 days or ( H and I ) indicated time points. G , ATP levels in osteoclasts (RLU, relative luminescence units; n = 4/group). H and I , protein levels (Western blot) in bone marrow macrophage cultures. To clarify Hem1 deletion in osteoclasts, the same images of Hem1 band are used. These are the same set of samples from the bone marrow macrophage cultures. J , osteoclasts developed in cultures of bone marrow macrophages from 6-month-old male C57BL/6 mice in the presence or the absence of imatinib (5 μM). Representative pictures ( left ), number ( middle ), and total area ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (triplicate cultures). K and L , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF and RANKL for ( K ) 5 or ( L ) 3 days in the presence or the absence of DPH (1 μM). K , number of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages. L , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis. Lines and error bars represent mean ± SD. p Values were determined with ( A – G ) Student’s t test, ( J ) one-way ANOVA, or ( K and L ) two-way ANOVA. DPH, 5-(1, 3-diaryl-1H-pyrazol-4-yl); Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Journal: The Journal of Biological Chemistry

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    doi: 10.1016/j.jbc.2022.102841

    Figure Lengend Snippet: Deletion of Hem1 attenuates respiration by suppressing c-Abl signaling. A – F , bone marrow macrophages from indicated genotypes were cultured with M-CSF and RANKL for 3 days. Different fractions of mitochondrial and nonmitochondrial respiration per 10,000 cells, in osteoclasts, were measured by Seahorse XF96 (n = 11–12 wells/group). G – I , bone marrow macrophages lacking Hem1 were cultured with M-CSF and RANKL for ( G ) 3 days or ( H and I ) indicated time points. G , ATP levels in osteoclasts (RLU, relative luminescence units; n = 4/group). H and I , protein levels (Western blot) in bone marrow macrophage cultures. To clarify Hem1 deletion in osteoclasts, the same images of Hem1 band are used. These are the same set of samples from the bone marrow macrophage cultures. J , osteoclasts developed in cultures of bone marrow macrophages from 6-month-old male C57BL/6 mice in the presence or the absence of imatinib (5 μM). Representative pictures ( left ), number ( middle ), and total area ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (triplicate cultures). K and L , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF and RANKL for ( K ) 5 or ( L ) 3 days in the presence or the absence of DPH (1 μM). K , number of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages. L , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis. Lines and error bars represent mean ± SD. p Values were determined with ( A – G ) Student’s t test, ( J ) one-way ANOVA, or ( K and L ) two-way ANOVA. DPH, 5-(1, 3-diaryl-1H-pyrazol-4-yl); Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

    Techniques: Cell Culture, Western Blot, Isolation, Knock-Out, Generated, Real-time Polymerase Chain Reaction

    Rescue of bone phenotype in Hem1-null mice by transplantation with wildtype hematopoietic cells. Hematopoietic stem cells from GFP + Lin − CD45 + wildtype mice efficiently repopulated the hematopoietic system of nonablated Hem1-deficient mice after bone marrow cell transplantation. Femurs and bone marrow were analyzed 10 weeks after transplantation (n = 5). A , schematic model of bone marrow transplantation. B , representative images of whole body ( left ) and longitudinal weight measurement ( right ). C , representative images of whole femur ( left ) and length measurement in 13-week-old male recipient Hem1 knockout mice and wildtype littermates ( right ). D , representative images of cortical bone at midshaft (scale bar represents 100 μm). E , cortical thickness and cortical perimeters at the midshaft. F , representative images of trabecular bone (scale bar represents 1 mm) and ( G ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. H , serum concentration of a collagen degradation product (CTx) by ELISA. I , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (triplicate cultures). J – M , bone marrow macrophages were isolated from 13-week-old recipient Hem1 knockout mice and wildtype littermates and cultured with M-CSF and RANKL for ( G , I , and J ) 5 days or ( K and M ) 3 days. J , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (n = 6). K , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis (triplicate cultures). L , representative pictures of DAPI ( blue , nuclei) and phalloidin ( red , actin rings) staining in osteoclast cultures (scale bar represents 500 μm). M , mRNA levels (quantitative PCR assay) of fusion markers during osteoclastogenesis (triplicate cultures). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Journal: The Journal of Biological Chemistry

    Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

    doi: 10.1016/j.jbc.2022.102841

    Figure Lengend Snippet: Rescue of bone phenotype in Hem1-null mice by transplantation with wildtype hematopoietic cells. Hematopoietic stem cells from GFP + Lin − CD45 + wildtype mice efficiently repopulated the hematopoietic system of nonablated Hem1-deficient mice after bone marrow cell transplantation. Femurs and bone marrow were analyzed 10 weeks after transplantation (n = 5). A , schematic model of bone marrow transplantation. B , representative images of whole body ( left ) and longitudinal weight measurement ( right ). C , representative images of whole femur ( left ) and length measurement in 13-week-old male recipient Hem1 knockout mice and wildtype littermates ( right ). D , representative images of cortical bone at midshaft (scale bar represents 100 μm). E , cortical thickness and cortical perimeters at the midshaft. F , representative images of trabecular bone (scale bar represents 1 mm) and ( G ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. H , serum concentration of a collagen degradation product (CTx) by ELISA. I , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (triplicate cultures). J – M , bone marrow macrophages were isolated from 13-week-old recipient Hem1 knockout mice and wildtype littermates and cultured with M-CSF and RANKL for ( G , I , and J ) 5 days or ( K and M ) 3 days. J , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (n = 6). K , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis (triplicate cultures). L , representative pictures of DAPI ( blue , nuclei) and phalloidin ( red , actin rings) staining in osteoclast cultures (scale bar represents 500 μm). M , mRNA levels (quantitative PCR assay) of fusion markers during osteoclastogenesis (triplicate cultures). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

    Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

    Techniques: Transplantation Assay, Knock-Out, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Cell Culture, Real-time Polymerase Chain Reaction