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rabbit pth1r polyclonal antibody  (Alomone Labs)


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    Alomone Labs rabbit pth1r polyclonal antibody
    A pool of <t>PTH1R</t> co‐localizes with Cav1 in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. (A) Double immunofluorescence staining of native PTH1R and Cav1 in MC3T3‐E1 osteoblasts. Immunofluorescence was assessed using an anti‐Cav1 primary antibody (mouse) and an anti‐PTH1R primary antibody (rabbit) for the PTH receptor. Alexa fluor 488 anti‐rabbit ( ) and Alexa fluor 568 anti‐mouse ( ) secondary IgG antibodies were used for PTH1R and Cav1, respectively. (B and C) Live cell imaging captures of MC3T3‐E1 osteoblastic (B) and MLO‐Y4 osteocytic (C) cells transfected with both and constructs for 24 h. Representative images were obtained by fluorescence confocal microscopy. Arrows depict microdomains where PTH1R and Cav1 co‐localize (A–C). White insets correspond to magnified regions of the cell images (A–C). (D) Co‐immunofluorescence stainings of MC3T3‐E1 cells transfected with both and constructs for 24 h and stimulated with PTHrP (1–37) 100 nM [5–15 min (min)], were assessed using an anti‐cavin primary antibody (rabbit). An Alexa fluor 633 anti‐rabbit ( ) was used as a secondary IgG antibody. 4′,6‐diamino‐ 2‐phenylindole dihydrochloride (DAPI) was used to stain the nucleus ( ). Representative overlaid images in blue, green, red and purple (merged) obtained by fluorescence confocal microscopy indicate discrete regions were PTH1R localizes in caveolae‐like microdomains of MC3T3‐E1 cells. (E) Percentages of cavin microdomains of the cell membrane expressing both and and stimulated or not with PTHrP (1–37) were evaluated using LASX Software. The results represent the mean ± SEM of three independent experiments and 4–10 cells evaluated for each experiment. SEM, standard error of the mean. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001.
    Rabbit Pth1r Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/apr-051/pmc12264463-68-32-36?v=Alomone+Labs
    Average 90 stars, based on 1 article reviews
    rabbit pth1r polyclonal antibody - by Bioz Stars, 2026-07
    90/100 stars

    Images

    1) Product Images from "Caveolin‐1 Regulates Parathyroid Hormone (PTH)‐Related Protein (PTHrP) Actions on PTH Receptor Type 1 in Bone Cells"

    Article Title: Caveolin‐1 Regulates Parathyroid Hormone (PTH)‐Related Protein (PTHrP) Actions on PTH Receptor Type 1 in Bone Cells

    Journal: Journal of Cellular Physiology

    doi: 10.1002/jcp.70067

    A pool of PTH1R co‐localizes with Cav1 in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. (A) Double immunofluorescence staining of native PTH1R and Cav1 in MC3T3‐E1 osteoblasts. Immunofluorescence was assessed using an anti‐Cav1 primary antibody (mouse) and an anti‐PTH1R primary antibody (rabbit) for the PTH receptor. Alexa fluor 488 anti‐rabbit ( ) and Alexa fluor 568 anti‐mouse ( ) secondary IgG antibodies were used for PTH1R and Cav1, respectively. (B and C) Live cell imaging captures of MC3T3‐E1 osteoblastic (B) and MLO‐Y4 osteocytic (C) cells transfected with both and constructs for 24 h. Representative images were obtained by fluorescence confocal microscopy. Arrows depict microdomains where PTH1R and Cav1 co‐localize (A–C). White insets correspond to magnified regions of the cell images (A–C). (D) Co‐immunofluorescence stainings of MC3T3‐E1 cells transfected with both and constructs for 24 h and stimulated with PTHrP (1–37) 100 nM [5–15 min (min)], were assessed using an anti‐cavin primary antibody (rabbit). An Alexa fluor 633 anti‐rabbit ( ) was used as a secondary IgG antibody. 4′,6‐diamino‐ 2‐phenylindole dihydrochloride (DAPI) was used to stain the nucleus ( ). Representative overlaid images in blue, green, red and purple (merged) obtained by fluorescence confocal microscopy indicate discrete regions were PTH1R localizes in caveolae‐like microdomains of MC3T3‐E1 cells. (E) Percentages of cavin microdomains of the cell membrane expressing both and and stimulated or not with PTHrP (1–37) were evaluated using LASX Software. The results represent the mean ± SEM of three independent experiments and 4–10 cells evaluated for each experiment. SEM, standard error of the mean. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001.
    Figure Legend Snippet: A pool of PTH1R co‐localizes with Cav1 in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. (A) Double immunofluorescence staining of native PTH1R and Cav1 in MC3T3‐E1 osteoblasts. Immunofluorescence was assessed using an anti‐Cav1 primary antibody (mouse) and an anti‐PTH1R primary antibody (rabbit) for the PTH receptor. Alexa fluor 488 anti‐rabbit ( ) and Alexa fluor 568 anti‐mouse ( ) secondary IgG antibodies were used for PTH1R and Cav1, respectively. (B and C) Live cell imaging captures of MC3T3‐E1 osteoblastic (B) and MLO‐Y4 osteocytic (C) cells transfected with both and constructs for 24 h. Representative images were obtained by fluorescence confocal microscopy. Arrows depict microdomains where PTH1R and Cav1 co‐localize (A–C). White insets correspond to magnified regions of the cell images (A–C). (D) Co‐immunofluorescence stainings of MC3T3‐E1 cells transfected with both and constructs for 24 h and stimulated with PTHrP (1–37) 100 nM [5–15 min (min)], were assessed using an anti‐cavin primary antibody (rabbit). An Alexa fluor 633 anti‐rabbit ( ) was used as a secondary IgG antibody. 4′,6‐diamino‐ 2‐phenylindole dihydrochloride (DAPI) was used to stain the nucleus ( ). Representative overlaid images in blue, green, red and purple (merged) obtained by fluorescence confocal microscopy indicate discrete regions were PTH1R localizes in caveolae‐like microdomains of MC3T3‐E1 cells. (E) Percentages of cavin microdomains of the cell membrane expressing both and and stimulated or not with PTHrP (1–37) were evaluated using LASX Software. The results represent the mean ± SEM of three independent experiments and 4–10 cells evaluated for each experiment. SEM, standard error of the mean. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001.

    Techniques Used: Double Immunofluorescence Staining, Immunofluorescence, Live Cell Imaging, Transfection, Construct, Fluorescence, Confocal Microscopy, Staining, Membrane, Expressing, Software

    Cav1 regulates PTHrP‐dependent expression of bone formation and remodeling genes in ex vivo cultured bones of aging mice and caveolae‐depleted mice and modulates alkaline phosphatase activity in MC3T3‐E1 osteoblastic cells. Femora of 2 and 18‐month‐old male (A) mice were cultured ex vivo for 6 days as described in materials and methods. (B) Femora of 2‐months old mice were treated with 5 µM MβCD and/or stimulated with 100 nM PTHrP (1–37) for 6 days following an intermittent cycle of stimulation [cycles of 24 h of inhibitor and/or PTHrP stimulation followed by 24 h of inhibitor‐ or peptide‐free medium incubation repeated three times during 6 days]. Expression of Cav1, PTH1R, Runx2, osteocalcin, alkaline phosphatase, OPG and RANKL was analyzed by real‐time PCR as described in materials and methods. The results represent the mean ± SEM ( n = 5–10 samples per experimental group). Interaction p values (I. p) between Cav1 and PTHrP actions on gene expression obtained by two‐way ANOVA are shown for each graph. Statistical significance: * p < 0.05. (C) Osteogenic differentatiation was assessed by analyzing alkaline phosphatase activity in MC3T3‐E1 osteoblasts transfected with specific siRNAs against Cav1 (KD Cav1) or with a scrambled siRNA sequence as control, incubated with ostegenic differentiation media and intermittent PTHrP (1–37) stimulation for 15 days as decribed in the methods section. The results represent the mean ± SEM of three independent experiments with two replicates for each experiment. Two‐way ANOVA analysis of Cav1 and PTHrP intertwined actions on alkaline phosphatase activity presented a significant interaction p value < 0.001.
    Figure Legend Snippet: Cav1 regulates PTHrP‐dependent expression of bone formation and remodeling genes in ex vivo cultured bones of aging mice and caveolae‐depleted mice and modulates alkaline phosphatase activity in MC3T3‐E1 osteoblastic cells. Femora of 2 and 18‐month‐old male (A) mice were cultured ex vivo for 6 days as described in materials and methods. (B) Femora of 2‐months old mice were treated with 5 µM MβCD and/or stimulated with 100 nM PTHrP (1–37) for 6 days following an intermittent cycle of stimulation [cycles of 24 h of inhibitor and/or PTHrP stimulation followed by 24 h of inhibitor‐ or peptide‐free medium incubation repeated three times during 6 days]. Expression of Cav1, PTH1R, Runx2, osteocalcin, alkaline phosphatase, OPG and RANKL was analyzed by real‐time PCR as described in materials and methods. The results represent the mean ± SEM ( n = 5–10 samples per experimental group). Interaction p values (I. p) between Cav1 and PTHrP actions on gene expression obtained by two‐way ANOVA are shown for each graph. Statistical significance: * p < 0.05. (C) Osteogenic differentatiation was assessed by analyzing alkaline phosphatase activity in MC3T3‐E1 osteoblasts transfected with specific siRNAs against Cav1 (KD Cav1) or with a scrambled siRNA sequence as control, incubated with ostegenic differentiation media and intermittent PTHrP (1–37) stimulation for 15 days as decribed in the methods section. The results represent the mean ± SEM of three independent experiments with two replicates for each experiment. Two‐way ANOVA analysis of Cav1 and PTHrP intertwined actions on alkaline phosphatase activity presented a significant interaction p value < 0.001.

    Techniques Used: Expressing, Ex Vivo, Cell Culture, Activity Assay, Incubation, Real-time Polymerase Chain Reaction, Gene Expression, Transfection, Sequencing, Control

    Cav1 reduces PTH1R mobility at membrane microdomains and decreases receptor internalization upon PTHrP stimulation in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. MC3T3‐E1 osteoblastic (A–C) and MLO‐Y4 osteocytic (D and E) cells were transfected with specific Cav1 or scrambled siRNAs for 24 h, and were subsequently transfected with . Alternatively some cells were transfected with both and for 24 h. (A) PTH1R diffusion along the cell membrane was determined by Fluorescence Recovery After Photobleaching (FRAP) as described in materials and methods. Analysis of FRAP in cells expressing native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) is shown. (B) Mobile or (C) immobile fractions of PTH1R in osteoblastic cells presenting native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) are shown. FRAP values obtained from MLO‐Y4 cells expressing native Cav1 or cells overexpressing Cav1 (D) or from Cav1 high‐ or low‐presenting membrane regions of MLO‐Y4 osteocytic cells transfected with Cav1 are shown (E). Representative immunoblots depicting immunoprecipitation of PTH1R and western blot of PTH1R (Top panel) or Cav1 (Bottom panel) are shown in MC3T3‐E1 cells (F). The input represents cell lysates before immunoprecipiation, IP represents the complex of beads and PTH1R antibodies after incubation and precipitation of the sample and Snt represents the supernatant obtained after removing the immunoprecipitation (IP) part of the sample. (G and H). MC3T3‐E1 cells were stimulated or not with PTHrP (1–37) for 5, 15 or 30 min and internalization analysis was performed as described in materials and methods. Briefly, PTH1R internalization after PTHrP stimulation was evaluated by “live cell imaging” for 30 min measuring the fluorescence intensity of areas at the cell membrane surface of MC3T3‐E1 osteoblasts during the period of study. (H) Representative images of PTH1R internalization for each condition are shown. GFP PTH1R is shown in green and in red (when present). Results in pcDNA3.1‐transfected cells closely resembled those of scrambled sRNA‐transfected cells and were collectively used as controls in the figures. The results represent the mean ± SEM of three independent experiments with five‐six replicates for each experiment (FRAP evaluations were assessed in 2–3 different regions for each replicate). Statistical significance: * p < 0.05, ** p < 0.01. SEM, standard error of the mean.
    Figure Legend Snippet: Cav1 reduces PTH1R mobility at membrane microdomains and decreases receptor internalization upon PTHrP stimulation in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. MC3T3‐E1 osteoblastic (A–C) and MLO‐Y4 osteocytic (D and E) cells were transfected with specific Cav1 or scrambled siRNAs for 24 h, and were subsequently transfected with . Alternatively some cells were transfected with both and for 24 h. (A) PTH1R diffusion along the cell membrane was determined by Fluorescence Recovery After Photobleaching (FRAP) as described in materials and methods. Analysis of FRAP in cells expressing native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) is shown. (B) Mobile or (C) immobile fractions of PTH1R in osteoblastic cells presenting native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) are shown. FRAP values obtained from MLO‐Y4 cells expressing native Cav1 or cells overexpressing Cav1 (D) or from Cav1 high‐ or low‐presenting membrane regions of MLO‐Y4 osteocytic cells transfected with Cav1 are shown (E). Representative immunoblots depicting immunoprecipitation of PTH1R and western blot of PTH1R (Top panel) or Cav1 (Bottom panel) are shown in MC3T3‐E1 cells (F). The input represents cell lysates before immunoprecipiation, IP represents the complex of beads and PTH1R antibodies after incubation and precipitation of the sample and Snt represents the supernatant obtained after removing the immunoprecipitation (IP) part of the sample. (G and H). MC3T3‐E1 cells were stimulated or not with PTHrP (1–37) for 5, 15 or 30 min and internalization analysis was performed as described in materials and methods. Briefly, PTH1R internalization after PTHrP stimulation was evaluated by “live cell imaging” for 30 min measuring the fluorescence intensity of areas at the cell membrane surface of MC3T3‐E1 osteoblasts during the period of study. (H) Representative images of PTH1R internalization for each condition are shown. GFP PTH1R is shown in green and in red (when present). Results in pcDNA3.1‐transfected cells closely resembled those of scrambled sRNA‐transfected cells and were collectively used as controls in the figures. The results represent the mean ± SEM of three independent experiments with five‐six replicates for each experiment (FRAP evaluations were assessed in 2–3 different regions for each replicate). Statistical significance: * p < 0.05, ** p < 0.01. SEM, standard error of the mean.

    Techniques Used: Membrane, Transfection, Diffusion-based Assay, Fluorescence, Expressing, Western Blot, Immunoprecipitation, Incubation, Live Cell Imaging

    Proposed mechanism of Cav1 regulation on PTH1R signaling, trafficking and regulation of gene expression. PTHrP increases PTH1R mobility at the cell membrane, triggers subsequent receptor internalization, stimulates Ca i 2+ release, promotes ERK1/2 kinase phosphorylation, increases AMPc levels and induces Runx2, ALP, and OCN gene expression. Cav1 knock down (KD) on the left, increases PTH1R mobility and internalization, causes increased Ca i 2+ release and ERK1/2 kinase phosphorylation and inhibits Runx2, ALP and OCN gene expression. Overexpression of Cav1 (OE Cav1) on the right, decreases PTH1R mobility and internalization, causes decreased Ca i 2+ release and increased cAMP accumulation and triggers higher although shorter overexpression of OCN and ALP gene expression. ALP, alkaline phosphatase; OCN, osteocalcin; PTH1R, parathyroid hormone receptor type 1; PTHrP, PTH‐related protein; Runx2, runt‐related transcription factor 2; SEM, standard error of the mean.
    Figure Legend Snippet: Proposed mechanism of Cav1 regulation on PTH1R signaling, trafficking and regulation of gene expression. PTHrP increases PTH1R mobility at the cell membrane, triggers subsequent receptor internalization, stimulates Ca i 2+ release, promotes ERK1/2 kinase phosphorylation, increases AMPc levels and induces Runx2, ALP, and OCN gene expression. Cav1 knock down (KD) on the left, increases PTH1R mobility and internalization, causes increased Ca i 2+ release and ERK1/2 kinase phosphorylation and inhibits Runx2, ALP and OCN gene expression. Overexpression of Cav1 (OE Cav1) on the right, decreases PTH1R mobility and internalization, causes decreased Ca i 2+ release and increased cAMP accumulation and triggers higher although shorter overexpression of OCN and ALP gene expression. ALP, alkaline phosphatase; OCN, osteocalcin; PTH1R, parathyroid hormone receptor type 1; PTHrP, PTH‐related protein; Runx2, runt‐related transcription factor 2; SEM, standard error of the mean.

    Techniques Used: Gene Expression, Membrane, Phospho-proteomics, Knockdown, Over Expression



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    rabbit pth1r polyclonal antibody  (Alomone Labs)
    90
    Alomone Labs rabbit pth1r polyclonal antibody
    A pool of <t>PTH1R</t> co‐localizes with Cav1 in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. (A) Double immunofluorescence staining of native PTH1R and Cav1 in MC3T3‐E1 osteoblasts. Immunofluorescence was assessed using an anti‐Cav1 primary antibody (mouse) and an anti‐PTH1R primary antibody (rabbit) for the PTH receptor. Alexa fluor 488 anti‐rabbit ( ) and Alexa fluor 568 anti‐mouse ( ) secondary IgG antibodies were used for PTH1R and Cav1, respectively. (B and C) Live cell imaging captures of MC3T3‐E1 osteoblastic (B) and MLO‐Y4 osteocytic (C) cells transfected with both and constructs for 24 h. Representative images were obtained by fluorescence confocal microscopy. Arrows depict microdomains where PTH1R and Cav1 co‐localize (A–C). White insets correspond to magnified regions of the cell images (A–C). (D) Co‐immunofluorescence stainings of MC3T3‐E1 cells transfected with both and constructs for 24 h and stimulated with PTHrP (1–37) 100 nM [5–15 min (min)], were assessed using an anti‐cavin primary antibody (rabbit). An Alexa fluor 633 anti‐rabbit ( ) was used as a secondary IgG antibody. 4′,6‐diamino‐ 2‐phenylindole dihydrochloride (DAPI) was used to stain the nucleus ( ). Representative overlaid images in blue, green, red and purple (merged) obtained by fluorescence confocal microscopy indicate discrete regions were PTH1R localizes in caveolae‐like microdomains of MC3T3‐E1 cells. (E) Percentages of cavin microdomains of the cell membrane expressing both and and stimulated or not with PTHrP (1–37) were evaluated using LASX Software. The results represent the mean ± SEM of three independent experiments and 4–10 cells evaluated for each experiment. SEM, standard error of the mean. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001.
    Rabbit Pth1r Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/apr-051/pmc12264463-68-32-36?v=Alomone+Labs
    Average 90 stars, based on 1 article reviews
    rabbit pth1r polyclonal antibody - by Bioz Stars, 2026-07
    90/100 stars
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    A pool of PTH1R co‐localizes with Cav1 in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. (A) Double immunofluorescence staining of native PTH1R and Cav1 in MC3T3‐E1 osteoblasts. Immunofluorescence was assessed using an anti‐Cav1 primary antibody (mouse) and an anti‐PTH1R primary antibody (rabbit) for the PTH receptor. Alexa fluor 488 anti‐rabbit ( ) and Alexa fluor 568 anti‐mouse ( ) secondary IgG antibodies were used for PTH1R and Cav1, respectively. (B and C) Live cell imaging captures of MC3T3‐E1 osteoblastic (B) and MLO‐Y4 osteocytic (C) cells transfected with both and constructs for 24 h. Representative images were obtained by fluorescence confocal microscopy. Arrows depict microdomains where PTH1R and Cav1 co‐localize (A–C). White insets correspond to magnified regions of the cell images (A–C). (D) Co‐immunofluorescence stainings of MC3T3‐E1 cells transfected with both and constructs for 24 h and stimulated with PTHrP (1–37) 100 nM [5–15 min (min)], were assessed using an anti‐cavin primary antibody (rabbit). An Alexa fluor 633 anti‐rabbit ( ) was used as a secondary IgG antibody. 4′,6‐diamino‐ 2‐phenylindole dihydrochloride (DAPI) was used to stain the nucleus ( ). Representative overlaid images in blue, green, red and purple (merged) obtained by fluorescence confocal microscopy indicate discrete regions were PTH1R localizes in caveolae‐like microdomains of MC3T3‐E1 cells. (E) Percentages of cavin microdomains of the cell membrane expressing both and and stimulated or not with PTHrP (1–37) were evaluated using LASX Software. The results represent the mean ± SEM of three independent experiments and 4–10 cells evaluated for each experiment. SEM, standard error of the mean. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001.

    Journal: Journal of Cellular Physiology

    Article Title: Caveolin‐1 Regulates Parathyroid Hormone (PTH)‐Related Protein (PTHrP) Actions on PTH Receptor Type 1 in Bone Cells

    doi: 10.1002/jcp.70067

    Figure Lengend Snippet: A pool of PTH1R co‐localizes with Cav1 in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. (A) Double immunofluorescence staining of native PTH1R and Cav1 in MC3T3‐E1 osteoblasts. Immunofluorescence was assessed using an anti‐Cav1 primary antibody (mouse) and an anti‐PTH1R primary antibody (rabbit) for the PTH receptor. Alexa fluor 488 anti‐rabbit ( ) and Alexa fluor 568 anti‐mouse ( ) secondary IgG antibodies were used for PTH1R and Cav1, respectively. (B and C) Live cell imaging captures of MC3T3‐E1 osteoblastic (B) and MLO‐Y4 osteocytic (C) cells transfected with both and constructs for 24 h. Representative images were obtained by fluorescence confocal microscopy. Arrows depict microdomains where PTH1R and Cav1 co‐localize (A–C). White insets correspond to magnified regions of the cell images (A–C). (D) Co‐immunofluorescence stainings of MC3T3‐E1 cells transfected with both and constructs for 24 h and stimulated with PTHrP (1–37) 100 nM [5–15 min (min)], were assessed using an anti‐cavin primary antibody (rabbit). An Alexa fluor 633 anti‐rabbit ( ) was used as a secondary IgG antibody. 4′,6‐diamino‐ 2‐phenylindole dihydrochloride (DAPI) was used to stain the nucleus ( ). Representative overlaid images in blue, green, red and purple (merged) obtained by fluorescence confocal microscopy indicate discrete regions were PTH1R localizes in caveolae‐like microdomains of MC3T3‐E1 cells. (E) Percentages of cavin microdomains of the cell membrane expressing both and and stimulated or not with PTHrP (1–37) were evaluated using LASX Software. The results represent the mean ± SEM of three independent experiments and 4–10 cells evaluated for each experiment. SEM, standard error of the mean. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001.

    Article Snippet: Nonspecific binding was blocked with 5% goat serum in bovine serum albumin (BSA) for 1 h, followed by overnight incubation with mouse Cav1 monoclonal antibody (Merck Millipore, Burlington, MA, USA) [dilution 1/1000], rabbit PTH1R polyclonal antibody (Alomone Labs, Jerusalem, Israel) [dilution 1/200] or rabbit Cavin/PTRF polyclonal antibody (Thermo Fischer Scientific) [dilution 1/200] at 4°C.

    Techniques: Double Immunofluorescence Staining, Immunofluorescence, Live Cell Imaging, Transfection, Construct, Fluorescence, Confocal Microscopy, Staining, Membrane, Expressing, Software

    Cav1 regulates PTHrP‐dependent expression of bone formation and remodeling genes in ex vivo cultured bones of aging mice and caveolae‐depleted mice and modulates alkaline phosphatase activity in MC3T3‐E1 osteoblastic cells. Femora of 2 and 18‐month‐old male (A) mice were cultured ex vivo for 6 days as described in materials and methods. (B) Femora of 2‐months old mice were treated with 5 µM MβCD and/or stimulated with 100 nM PTHrP (1–37) for 6 days following an intermittent cycle of stimulation [cycles of 24 h of inhibitor and/or PTHrP stimulation followed by 24 h of inhibitor‐ or peptide‐free medium incubation repeated three times during 6 days]. Expression of Cav1, PTH1R, Runx2, osteocalcin, alkaline phosphatase, OPG and RANKL was analyzed by real‐time PCR as described in materials and methods. The results represent the mean ± SEM ( n = 5–10 samples per experimental group). Interaction p values (I. p) between Cav1 and PTHrP actions on gene expression obtained by two‐way ANOVA are shown for each graph. Statistical significance: * p < 0.05. (C) Osteogenic differentatiation was assessed by analyzing alkaline phosphatase activity in MC3T3‐E1 osteoblasts transfected with specific siRNAs against Cav1 (KD Cav1) or with a scrambled siRNA sequence as control, incubated with ostegenic differentiation media and intermittent PTHrP (1–37) stimulation for 15 days as decribed in the methods section. The results represent the mean ± SEM of three independent experiments with two replicates for each experiment. Two‐way ANOVA analysis of Cav1 and PTHrP intertwined actions on alkaline phosphatase activity presented a significant interaction p value < 0.001.

    Journal: Journal of Cellular Physiology

    Article Title: Caveolin‐1 Regulates Parathyroid Hormone (PTH)‐Related Protein (PTHrP) Actions on PTH Receptor Type 1 in Bone Cells

    doi: 10.1002/jcp.70067

    Figure Lengend Snippet: Cav1 regulates PTHrP‐dependent expression of bone formation and remodeling genes in ex vivo cultured bones of aging mice and caveolae‐depleted mice and modulates alkaline phosphatase activity in MC3T3‐E1 osteoblastic cells. Femora of 2 and 18‐month‐old male (A) mice were cultured ex vivo for 6 days as described in materials and methods. (B) Femora of 2‐months old mice were treated with 5 µM MβCD and/or stimulated with 100 nM PTHrP (1–37) for 6 days following an intermittent cycle of stimulation [cycles of 24 h of inhibitor and/or PTHrP stimulation followed by 24 h of inhibitor‐ or peptide‐free medium incubation repeated three times during 6 days]. Expression of Cav1, PTH1R, Runx2, osteocalcin, alkaline phosphatase, OPG and RANKL was analyzed by real‐time PCR as described in materials and methods. The results represent the mean ± SEM ( n = 5–10 samples per experimental group). Interaction p values (I. p) between Cav1 and PTHrP actions on gene expression obtained by two‐way ANOVA are shown for each graph. Statistical significance: * p < 0.05. (C) Osteogenic differentatiation was assessed by analyzing alkaline phosphatase activity in MC3T3‐E1 osteoblasts transfected with specific siRNAs against Cav1 (KD Cav1) or with a scrambled siRNA sequence as control, incubated with ostegenic differentiation media and intermittent PTHrP (1–37) stimulation for 15 days as decribed in the methods section. The results represent the mean ± SEM of three independent experiments with two replicates for each experiment. Two‐way ANOVA analysis of Cav1 and PTHrP intertwined actions on alkaline phosphatase activity presented a significant interaction p value < 0.001.

    Article Snippet: Nonspecific binding was blocked with 5% goat serum in bovine serum albumin (BSA) for 1 h, followed by overnight incubation with mouse Cav1 monoclonal antibody (Merck Millipore, Burlington, MA, USA) [dilution 1/1000], rabbit PTH1R polyclonal antibody (Alomone Labs, Jerusalem, Israel) [dilution 1/200] or rabbit Cavin/PTRF polyclonal antibody (Thermo Fischer Scientific) [dilution 1/200] at 4°C.

    Techniques: Expressing, Ex Vivo, Cell Culture, Activity Assay, Incubation, Real-time Polymerase Chain Reaction, Gene Expression, Transfection, Sequencing, Control

    Cav1 reduces PTH1R mobility at membrane microdomains and decreases receptor internalization upon PTHrP stimulation in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. MC3T3‐E1 osteoblastic (A–C) and MLO‐Y4 osteocytic (D and E) cells were transfected with specific Cav1 or scrambled siRNAs for 24 h, and were subsequently transfected with . Alternatively some cells were transfected with both and for 24 h. (A) PTH1R diffusion along the cell membrane was determined by Fluorescence Recovery After Photobleaching (FRAP) as described in materials and methods. Analysis of FRAP in cells expressing native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) is shown. (B) Mobile or (C) immobile fractions of PTH1R in osteoblastic cells presenting native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) are shown. FRAP values obtained from MLO‐Y4 cells expressing native Cav1 or cells overexpressing Cav1 (D) or from Cav1 high‐ or low‐presenting membrane regions of MLO‐Y4 osteocytic cells transfected with Cav1 are shown (E). Representative immunoblots depicting immunoprecipitation of PTH1R and western blot of PTH1R (Top panel) or Cav1 (Bottom panel) are shown in MC3T3‐E1 cells (F). The input represents cell lysates before immunoprecipiation, IP represents the complex of beads and PTH1R antibodies after incubation and precipitation of the sample and Snt represents the supernatant obtained after removing the immunoprecipitation (IP) part of the sample. (G and H). MC3T3‐E1 cells were stimulated or not with PTHrP (1–37) for 5, 15 or 30 min and internalization analysis was performed as described in materials and methods. Briefly, PTH1R internalization after PTHrP stimulation was evaluated by “live cell imaging” for 30 min measuring the fluorescence intensity of areas at the cell membrane surface of MC3T3‐E1 osteoblasts during the period of study. (H) Representative images of PTH1R internalization for each condition are shown. GFP PTH1R is shown in green and in red (when present). Results in pcDNA3.1‐transfected cells closely resembled those of scrambled sRNA‐transfected cells and were collectively used as controls in the figures. The results represent the mean ± SEM of three independent experiments with five‐six replicates for each experiment (FRAP evaluations were assessed in 2–3 different regions for each replicate). Statistical significance: * p < 0.05, ** p < 0.01. SEM, standard error of the mean.

    Journal: Journal of Cellular Physiology

    Article Title: Caveolin‐1 Regulates Parathyroid Hormone (PTH)‐Related Protein (PTHrP) Actions on PTH Receptor Type 1 in Bone Cells

    doi: 10.1002/jcp.70067

    Figure Lengend Snippet: Cav1 reduces PTH1R mobility at membrane microdomains and decreases receptor internalization upon PTHrP stimulation in MC3T3‐E1 osteoblastic and MLO‐Y4 osteocytic cells. MC3T3‐E1 osteoblastic (A–C) and MLO‐Y4 osteocytic (D and E) cells were transfected with specific Cav1 or scrambled siRNAs for 24 h, and were subsequently transfected with . Alternatively some cells were transfected with both and for 24 h. (A) PTH1R diffusion along the cell membrane was determined by Fluorescence Recovery After Photobleaching (FRAP) as described in materials and methods. Analysis of FRAP in cells expressing native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) is shown. (B) Mobile or (C) immobile fractions of PTH1R in osteoblastic cells presenting native Cav1, cells transfected with Cav1 siRNA (KD Cav1), or cells overexpressing Cav1 (OE Cav1) are shown. FRAP values obtained from MLO‐Y4 cells expressing native Cav1 or cells overexpressing Cav1 (D) or from Cav1 high‐ or low‐presenting membrane regions of MLO‐Y4 osteocytic cells transfected with Cav1 are shown (E). Representative immunoblots depicting immunoprecipitation of PTH1R and western blot of PTH1R (Top panel) or Cav1 (Bottom panel) are shown in MC3T3‐E1 cells (F). The input represents cell lysates before immunoprecipiation, IP represents the complex of beads and PTH1R antibodies after incubation and precipitation of the sample and Snt represents the supernatant obtained after removing the immunoprecipitation (IP) part of the sample. (G and H). MC3T3‐E1 cells were stimulated or not with PTHrP (1–37) for 5, 15 or 30 min and internalization analysis was performed as described in materials and methods. Briefly, PTH1R internalization after PTHrP stimulation was evaluated by “live cell imaging” for 30 min measuring the fluorescence intensity of areas at the cell membrane surface of MC3T3‐E1 osteoblasts during the period of study. (H) Representative images of PTH1R internalization for each condition are shown. GFP PTH1R is shown in green and in red (when present). Results in pcDNA3.1‐transfected cells closely resembled those of scrambled sRNA‐transfected cells and were collectively used as controls in the figures. The results represent the mean ± SEM of three independent experiments with five‐six replicates for each experiment (FRAP evaluations were assessed in 2–3 different regions for each replicate). Statistical significance: * p < 0.05, ** p < 0.01. SEM, standard error of the mean.

    Article Snippet: Nonspecific binding was blocked with 5% goat serum in bovine serum albumin (BSA) for 1 h, followed by overnight incubation with mouse Cav1 monoclonal antibody (Merck Millipore, Burlington, MA, USA) [dilution 1/1000], rabbit PTH1R polyclonal antibody (Alomone Labs, Jerusalem, Israel) [dilution 1/200] or rabbit Cavin/PTRF polyclonal antibody (Thermo Fischer Scientific) [dilution 1/200] at 4°C.

    Techniques: Membrane, Transfection, Diffusion-based Assay, Fluorescence, Expressing, Western Blot, Immunoprecipitation, Incubation, Live Cell Imaging

    Proposed mechanism of Cav1 regulation on PTH1R signaling, trafficking and regulation of gene expression. PTHrP increases PTH1R mobility at the cell membrane, triggers subsequent receptor internalization, stimulates Ca i 2+ release, promotes ERK1/2 kinase phosphorylation, increases AMPc levels and induces Runx2, ALP, and OCN gene expression. Cav1 knock down (KD) on the left, increases PTH1R mobility and internalization, causes increased Ca i 2+ release and ERK1/2 kinase phosphorylation and inhibits Runx2, ALP and OCN gene expression. Overexpression of Cav1 (OE Cav1) on the right, decreases PTH1R mobility and internalization, causes decreased Ca i 2+ release and increased cAMP accumulation and triggers higher although shorter overexpression of OCN and ALP gene expression. ALP, alkaline phosphatase; OCN, osteocalcin; PTH1R, parathyroid hormone receptor type 1; PTHrP, PTH‐related protein; Runx2, runt‐related transcription factor 2; SEM, standard error of the mean.

    Journal: Journal of Cellular Physiology

    Article Title: Caveolin‐1 Regulates Parathyroid Hormone (PTH)‐Related Protein (PTHrP) Actions on PTH Receptor Type 1 in Bone Cells

    doi: 10.1002/jcp.70067

    Figure Lengend Snippet: Proposed mechanism of Cav1 regulation on PTH1R signaling, trafficking and regulation of gene expression. PTHrP increases PTH1R mobility at the cell membrane, triggers subsequent receptor internalization, stimulates Ca i 2+ release, promotes ERK1/2 kinase phosphorylation, increases AMPc levels and induces Runx2, ALP, and OCN gene expression. Cav1 knock down (KD) on the left, increases PTH1R mobility and internalization, causes increased Ca i 2+ release and ERK1/2 kinase phosphorylation and inhibits Runx2, ALP and OCN gene expression. Overexpression of Cav1 (OE Cav1) on the right, decreases PTH1R mobility and internalization, causes decreased Ca i 2+ release and increased cAMP accumulation and triggers higher although shorter overexpression of OCN and ALP gene expression. ALP, alkaline phosphatase; OCN, osteocalcin; PTH1R, parathyroid hormone receptor type 1; PTHrP, PTH‐related protein; Runx2, runt‐related transcription factor 2; SEM, standard error of the mean.

    Article Snippet: Nonspecific binding was blocked with 5% goat serum in bovine serum albumin (BSA) for 1 h, followed by overnight incubation with mouse Cav1 monoclonal antibody (Merck Millipore, Burlington, MA, USA) [dilution 1/1000], rabbit PTH1R polyclonal antibody (Alomone Labs, Jerusalem, Israel) [dilution 1/200] or rabbit Cavin/PTRF polyclonal antibody (Thermo Fischer Scientific) [dilution 1/200] at 4°C.

    Techniques: Gene Expression, Membrane, Phospho-proteomics, Knockdown, Over Expression