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Cell Signaling Technology Inc rabbit polyclonal anti total camkii

( A ) FSS causes the rapid loss of sclerostin protein through a number of molecular mediators. ( B ) Ocy454 cells (n = 3–4) or ( C ) UMR106 cells (n = 3) were exposed to 1 min of FSS at 4 dynes/cm 2 and lysed 5 min post-flow. Western blots were probed for sclerostin, GAPDH, pCaMKII, and total <t>CaMKII.</t> ( D ) Sixteen week old female C57Bl/6 mice were ulnar loaded (1800 με, 90 s, 2 Hz), cortical osteocyte-enriched lysates isolated 5 min post-load, and western blots probed for sclerostin (n = 10 mice), pCaMKII, and total CaMKII (n = 5 mice). Sclerostin abundance relative to the loading control or pCaMKII relative to total CaMKII was quantified. ( E ) Ocy454 cells with endogenous sclerostin (n = 2), ( F ) UMR106 cells with endogenous sclerostin (n = 4), or ( G ) Ocy454 cells transfected with Myc-tagged sclerostin (n = 1) were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed at the indicated times post-flow. Western blots were probed for sclerostin and β-actin. A representative time course is shown for each. Sclerostin abundance relative to the loading control was quantified. For each antibody, western blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-tests ( B–D ).

Rabbit Polyclonal Anti Total Camkii, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 459 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein"

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

Journal: eLife

doi: 10.7554/eLife.64393

Figure Legend Snippet: ( A ) FSS causes the rapid loss of sclerostin protein through a number of molecular mediators. ( B ) Ocy454 cells (n = 3–4) or ( C ) UMR106 cells (n = 3) were exposed to 1 min of FSS at 4 dynes/cm 2 and lysed 5 min post-flow. Western blots were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII. ( D ) Sixteen week old female C57Bl/6 mice were ulnar loaded (1800 με, 90 s, 2 Hz), cortical osteocyte-enriched lysates isolated 5 min post-load, and western blots probed for sclerostin (n = 10 mice), pCaMKII, and total CaMKII (n = 5 mice). Sclerostin abundance relative to the loading control or pCaMKII relative to total CaMKII was quantified. ( E ) Ocy454 cells with endogenous sclerostin (n = 2), ( F ) UMR106 cells with endogenous sclerostin (n = 4), or ( G ) Ocy454 cells transfected with Myc-tagged sclerostin (n = 1) were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed at the indicated times post-flow. Western blots were probed for sclerostin and β-actin. A representative time course is shown for each. Sclerostin abundance relative to the loading control was quantified. For each antibody, western blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-tests ( B–D ).

Techniques Used: Western Blot, Isolation, Control, Transfection, Two Tailed Test

( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for sclerostin and β-actin (n = 2–3). ( B ) Dissected tibiae flushed of marrow were treated with vehicle or PTH (1–34) (10 nM) for 30 min ex vivo and homogenized. Western blots were probed for sclerostin and β-actin (n = 6 mice). ( C ) Ocy454 cells were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for pCaMKII and total CaMKII (n = 6–8). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( A, C ) or unpaired two-tailed t-test ( B ).

Figure Legend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for sclerostin and β-actin (n = 2–3). ( B ) Dissected tibiae flushed of marrow were treated with vehicle or PTH (1–34) (10 nM) for 30 min ex vivo and homogenized. Western blots were probed for sclerostin and β-actin (n = 6 mice). ( C ) Ocy454 cells were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for pCaMKII and total CaMKII (n = 6–8). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( A, C ) or unpaired two-tailed t-test ( B ).

Techniques Used: Transfection, Western Blot, Ex Vivo, Two Tailed Test

( A ) Endogenous sclerostin (top) and GFP-tagged sclerostin (bottom) form discrete puncta in Ocy454 cells. ( B ) Ocy454 cells were transfected with GFP-sclerostin, and lysosomes were visualized with Lysotracker (1 mM, 1 hr) or siR-Lysosome (1 μM, 4 hr). Scale bar represents 10 μm. ( C ) Ocy454 cells were stained for endogenous sclerostin and either p62/sequestosome-1 or Rab27a to evaluate co-localization with these lysosome-associated proteins. ( D ) Ocy454 cells were exposed to 1 min of FSS at 4 dynes/cm 2 , lysed immediately post-flow, and western blotted for p62/sequestosome-1, β-actin, and LC3 (n = 4). ( E ) Ocy454 cells were treated with PTH (1–34) (10 nM) for 5 min, lysed, and western blotted for p62/sequestosome-1 and β-actin (n = 4) and LC3 (n = 8). ( F ) UMR106 cells were subjected to FSS for 5 min, then Magic Red Cathepsin B was applied for 10 min, fixed, and imaged to assess lysosome activity (n = 9). ( G ) Ocy454 cells were treated with DMSO or KN-93 (10 μM) to inhibit CaMKII for 1 hr prior to FSS at 4 dynes/cm 2 for 5 min before lysing immediately after FSS. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). ( H ) Ocy454 cells were transfected with a plasmid expressing either GFP or dominant negative CaMKII T286A prior to treatment with PTH (1–34) (10 nM) for 30 min. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-test ( D–F ) or by two-way ANOVA with Holm–Sidak post hoc correction ( G, H ).

Figure Legend Snippet: ( A ) Endogenous sclerostin (top) and GFP-tagged sclerostin (bottom) form discrete puncta in Ocy454 cells. ( B ) Ocy454 cells were transfected with GFP-sclerostin, and lysosomes were visualized with Lysotracker (1 mM, 1 hr) or siR-Lysosome (1 μM, 4 hr). Scale bar represents 10 μm. ( C ) Ocy454 cells were stained for endogenous sclerostin and either p62/sequestosome-1 or Rab27a to evaluate co-localization with these lysosome-associated proteins. ( D ) Ocy454 cells were exposed to 1 min of FSS at 4 dynes/cm 2 , lysed immediately post-flow, and western blotted for p62/sequestosome-1, β-actin, and LC3 (n = 4). ( E ) Ocy454 cells were treated with PTH (1–34) (10 nM) for 5 min, lysed, and western blotted for p62/sequestosome-1 and β-actin (n = 4) and LC3 (n = 8). ( F ) UMR106 cells were subjected to FSS for 5 min, then Magic Red Cathepsin B was applied for 10 min, fixed, and imaged to assess lysosome activity (n = 9). ( G ) Ocy454 cells were treated with DMSO or KN-93 (10 μM) to inhibit CaMKII for 1 hr prior to FSS at 4 dynes/cm 2 for 5 min before lysing immediately after FSS. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). ( H ) Ocy454 cells were transfected with a plasmid expressing either GFP or dominant negative CaMKII T286A prior to treatment with PTH (1–34) (10 nM) for 30 min. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-test ( D–F ) or by two-way ANOVA with Holm–Sidak post hoc correction ( G, H ).

Techniques Used: Transfection, Staining, Western Blot, Activity Assay, Plasmid Preparation, Expressing, Dominant Negative Mutation, Two Tailed Test

( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle (water) or 10 μM SNAP, a nitric oxide donor, and lysed after 5 min. Western blots were probed for sclerostin, α-tubulin, pCaMKII, and total CaMKII (n = 3). For each antibody, blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. ( B ) Ocy454 cells transfected with myc-tagged sclerostin were treated with DMSO or bafilomycin A1 (100 nM) to inhibit lysosomal degradation, for 30 min, then treated with SNAP, a nitric oxide donor, for 5 min and lysed. Western blots were probed for sclerostin and α-tubulin (n = 2–3). ( C ) UMR106 cells were treated with vehicle or L-NAME (1 mM) to inhibit nitric oxide synthases (NOSs) for 1 hr and then exposed to 1 or 5 min of FSS. Lysates from cells exposed to 1 min of FSS were probed for p62/sequestosome-1 and α-tubulin abundance and lysates from cells exposed to 5 min of sclerostin were probed for sclerostin and α-tubulin abundance (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01, ***p<0.001 by unpaired two-tailed t-test ( A ) or two-way ANOVA with Holm–Sidak post hoc test ( B , C ).

Figure Legend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle (water) or 10 μM SNAP, a nitric oxide donor, and lysed after 5 min. Western blots were probed for sclerostin, α-tubulin, pCaMKII, and total CaMKII (n = 3). For each antibody, blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. ( B ) Ocy454 cells transfected with myc-tagged sclerostin were treated with DMSO or bafilomycin A1 (100 nM) to inhibit lysosomal degradation, for 30 min, then treated with SNAP, a nitric oxide donor, for 5 min and lysed. Western blots were probed for sclerostin and α-tubulin (n = 2–3). ( C ) UMR106 cells were treated with vehicle or L-NAME (1 mM) to inhibit nitric oxide synthases (NOSs) for 1 hr and then exposed to 1 or 5 min of FSS. Lysates from cells exposed to 1 min of FSS were probed for p62/sequestosome-1 and α-tubulin abundance and lysates from cells exposed to 5 min of sclerostin were probed for sclerostin and α-tubulin abundance (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01, ***p<0.001 by unpaired two-tailed t-test ( A ) or two-way ANOVA with Holm–Sidak post hoc test ( B , C ).

Techniques Used: Transfection, Western Blot, Two Tailed Test

( A ) UMR106 cells transfected with KillerRed imaged before and after stimulation with LED light. DCF was used to track ROS production. ( B ) UMR106 cells transfected with KillerRed were stimulated with LED light for 5 min and lysed 5 min after. Westerns were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII.

Figure Legend Snippet: ( A ) UMR106 cells transfected with KillerRed imaged before and after stimulation with LED light. DCF was used to track ROS production. ( B ) UMR106 cells transfected with KillerRed were stimulated with LED light for 5 min and lysed 5 min after. Westerns were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII.

Techniques Used: Transfection

( A ) Fifteen week old male C57Bl/6 mice treated with vehicle (saline + 4% DMSO, n = 7 mice) or bafilomycin A1 (1 mg/kg, n = 7 mice) to inhibit lysosomal degradation were forearm loaded (2000 με, 90 s, 2 Hz) and labeled with calcein and alizarin red at the indicated times for dynamic histomorphometry. Representative periosteal double labeling are shown. ( B ) Periosteal bone formation rate (Ps.BFR) and ( C ) periosteal mineral apposition rate (Ps.MAR) were calculated. ( D ) Fourteen to 17 week old male and female C57Bl/6 mice treated with vehicle (saline + 4% DMSO, i.p., n = 14) or bafilomycin A1 (1 mg/kg in saline + 4% DMSO, i.p., n = 12 mice) to inhibit lysosomal degradation were treated 2 hr prior to ulnar loading (2000 με, 90 s, 2 Hz). Non-loaded and loaded limbs were isolated 5 min post-load, and western blots were probed for sclerostin and β-actin. Vehicle data is duplicated in as all animals were run and processed together. ( E ) Human iPSC-derived osteoblasts from either control (non-diseased) or Gaucher disease patients were treated with vehicle or recombinant glucocerebrosidase (rGCase, 0.24 U/mL) for 5 days, then lysed for western blotting. Western blots were probed for sclerostin and GAPDH (n = 3 independent patient-derived iPSC lines/group). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( B , C , and E ) or Kruskal–Wallis with Dunn’s post hoc correction ( D ). ( F ) FSS causes the rapid degradation of sclerostin protein by the lysosome through a number of molecular mediators. PTH, converging with this FSS mechano-transduction pathway at CaMKII, also mediates the lysosomal degradation of sclerostin protein. Icons outlined red are molecular mechanisms controlling sclerostin abundance that have been described within this manuscript that integrate into our previously described mechano-transduction cascade. Osteoanabolic stimuli, working through reactive oxygen (ROS) and reactive nitrogen species (RNS), direct sclerostin to the lysosome for degradation. This results in reduced sclerostin to allow for bone formation. PM: plasma membrane; ROS: reactive oxygen species; NO: nitric oxide.

Figure Legend Snippet: ( A ) Fifteen week old male C57Bl/6 mice treated with vehicle (saline + 4% DMSO, n = 7 mice) or bafilomycin A1 (1 mg/kg, n = 7 mice) to inhibit lysosomal degradation were forearm loaded (2000 με, 90 s, 2 Hz) and labeled with calcein and alizarin red at the indicated times for dynamic histomorphometry. Representative periosteal double labeling are shown. ( B ) Periosteal bone formation rate (Ps.BFR) and ( C ) periosteal mineral apposition rate (Ps.MAR) were calculated. ( D ) Fourteen to 17 week old male and female C57Bl/6 mice treated with vehicle (saline + 4% DMSO, i.p., n = 14) or bafilomycin A1 (1 mg/kg in saline + 4% DMSO, i.p., n = 12 mice) to inhibit lysosomal degradation were treated 2 hr prior to ulnar loading (2000 με, 90 s, 2 Hz). Non-loaded and loaded limbs were isolated 5 min post-load, and western blots were probed for sclerostin and β-actin. Vehicle data is duplicated in as all animals were run and processed together. ( E ) Human iPSC-derived osteoblasts from either control (non-diseased) or Gaucher disease patients were treated with vehicle or recombinant glucocerebrosidase (rGCase, 0.24 U/mL) for 5 days, then lysed for western blotting. Western blots were probed for sclerostin and GAPDH (n = 3 independent patient-derived iPSC lines/group). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( B , C , and E ) or Kruskal–Wallis with Dunn’s post hoc correction ( D ). ( F ) FSS causes the rapid degradation of sclerostin protein by the lysosome through a number of molecular mediators. PTH, converging with this FSS mechano-transduction pathway at CaMKII, also mediates the lysosomal degradation of sclerostin protein. Icons outlined red are molecular mechanisms controlling sclerostin abundance that have been described within this manuscript that integrate into our previously described mechano-transduction cascade. Osteoanabolic stimuli, working through reactive oxygen (ROS) and reactive nitrogen species (RNS), direct sclerostin to the lysosome for degradation. This results in reduced sclerostin to allow for bone formation. PM: plasma membrane; ROS: reactive oxygen species; NO: nitric oxide.

Techniques Used: Saline, Labeling, Isolation, Western Blot, Derivative Assay, Control, Recombinant, Transduction, Clinical Proteomics, Membrane

Figure Legend Snippet:

Techniques Used: Transfection, Construct, Sequencing, In Vitro, In Vivo, Software, Staining

Image Search Results

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) FSS causes the rapid loss of sclerostin protein through a number of molecular mediators. ( B ) Ocy454 cells (n = 3–4) or ( C ) UMR106 cells (n = 3) were exposed to 1 min of FSS at 4 dynes/cm 2 and lysed 5 min post-flow. Western blots were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII. ( D ) Sixteen week old female C57Bl/6 mice were ulnar loaded (1800 με, 90 s, 2 Hz), cortical osteocyte-enriched lysates isolated 5 min post-load, and western blots probed for sclerostin (n = 10 mice), pCaMKII, and total CaMKII (n = 5 mice). Sclerostin abundance relative to the loading control or pCaMKII relative to total CaMKII was quantified. ( E ) Ocy454 cells with endogenous sclerostin (n = 2), ( F ) UMR106 cells with endogenous sclerostin (n = 4), or ( G ) Ocy454 cells transfected with Myc-tagged sclerostin (n = 1) were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed at the indicated times post-flow. Western blots were probed for sclerostin and β-actin. A representative time course is shown for each. Sclerostin abundance relative to the loading control was quantified. For each antibody, western blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-tests ( B–D ).

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Western Blot, Isolation, Control, Transfection, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for sclerostin and β-actin (n = 2–3). ( B ) Dissected tibiae flushed of marrow were treated with vehicle or PTH (1–34) (10 nM) for 30 min ex vivo and homogenized. Western blots were probed for sclerostin and β-actin (n = 6 mice). ( C ) Ocy454 cells were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for pCaMKII and total CaMKII (n = 6–8). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( A, C ) or unpaired two-tailed t-test ( B ).

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Transfection, Western Blot, Ex Vivo, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Endogenous sclerostin (top) and GFP-tagged sclerostin (bottom) form discrete puncta in Ocy454 cells. ( B ) Ocy454 cells were transfected with GFP-sclerostin, and lysosomes were visualized with Lysotracker (1 mM, 1 hr) or siR-Lysosome (1 μM, 4 hr). Scale bar represents 10 μm. ( C ) Ocy454 cells were stained for endogenous sclerostin and either p62/sequestosome-1 or Rab27a to evaluate co-localization with these lysosome-associated proteins. ( D ) Ocy454 cells were exposed to 1 min of FSS at 4 dynes/cm 2 , lysed immediately post-flow, and western blotted for p62/sequestosome-1, β-actin, and LC3 (n = 4). ( E ) Ocy454 cells were treated with PTH (1–34) (10 nM) for 5 min, lysed, and western blotted for p62/sequestosome-1 and β-actin (n = 4) and LC3 (n = 8). ( F ) UMR106 cells were subjected to FSS for 5 min, then Magic Red Cathepsin B was applied for 10 min, fixed, and imaged to assess lysosome activity (n = 9). ( G ) Ocy454 cells were treated with DMSO or KN-93 (10 μM) to inhibit CaMKII for 1 hr prior to FSS at 4 dynes/cm 2 for 5 min before lysing immediately after FSS. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). ( H ) Ocy454 cells were transfected with a plasmid expressing either GFP or dominant negative CaMKII T286A prior to treatment with PTH (1–34) (10 nM) for 30 min. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-test ( D–F ) or by two-way ANOVA with Holm–Sidak post hoc correction ( G, H ).

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Transfection, Staining, Western Blot, Activity Assay, Plasmid Preparation, Expressing, Dominant Negative Mutation, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle (water) or 10 μM SNAP, a nitric oxide donor, and lysed after 5 min. Western blots were probed for sclerostin, α-tubulin, pCaMKII, and total CaMKII (n = 3). For each antibody, blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. ( B ) Ocy454 cells transfected with myc-tagged sclerostin were treated with DMSO or bafilomycin A1 (100 nM) to inhibit lysosomal degradation, for 30 min, then treated with SNAP, a nitric oxide donor, for 5 min and lysed. Western blots were probed for sclerostin and α-tubulin (n = 2–3). ( C ) UMR106 cells were treated with vehicle or L-NAME (1 mM) to inhibit nitric oxide synthases (NOSs) for 1 hr and then exposed to 1 or 5 min of FSS. Lysates from cells exposed to 1 min of FSS were probed for p62/sequestosome-1 and α-tubulin abundance and lysates from cells exposed to 5 min of sclerostin were probed for sclerostin and α-tubulin abundance (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01, ***p<0.001 by unpaired two-tailed t-test ( A ) or two-way ANOVA with Holm–Sidak post hoc test ( B , C ).

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Transfection, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) UMR106 cells transfected with KillerRed imaged before and after stimulation with LED light. DCF was used to track ROS production. ( B ) UMR106 cells transfected with KillerRed were stimulated with LED light for 5 min and lysed 5 min after. Westerns were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII.

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Transfection

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Fifteen week old male C57Bl/6 mice treated with vehicle (saline + 4% DMSO, n = 7 mice) or bafilomycin A1 (1 mg/kg, n = 7 mice) to inhibit lysosomal degradation were forearm loaded (2000 με, 90 s, 2 Hz) and labeled with calcein and alizarin red at the indicated times for dynamic histomorphometry. Representative periosteal double labeling are shown. ( B ) Periosteal bone formation rate (Ps.BFR) and ( C ) periosteal mineral apposition rate (Ps.MAR) were calculated. ( D ) Fourteen to 17 week old male and female C57Bl/6 mice treated with vehicle (saline + 4% DMSO, i.p., n = 14) or bafilomycin A1 (1 mg/kg in saline + 4% DMSO, i.p., n = 12 mice) to inhibit lysosomal degradation were treated 2 hr prior to ulnar loading (2000 με, 90 s, 2 Hz). Non-loaded and loaded limbs were isolated 5 min post-load, and western blots were probed for sclerostin and β-actin. Vehicle data is duplicated in as all animals were run and processed together. ( E ) Human iPSC-derived osteoblasts from either control (non-diseased) or Gaucher disease patients were treated with vehicle or recombinant glucocerebrosidase (rGCase, 0.24 U/mL) for 5 days, then lysed for western blotting. Western blots were probed for sclerostin and GAPDH (n = 3 independent patient-derived iPSC lines/group). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( B , C , and E ) or Kruskal–Wallis with Dunn’s post hoc correction ( D ). ( F ) FSS causes the rapid degradation of sclerostin protein by the lysosome through a number of molecular mediators. PTH, converging with this FSS mechano-transduction pathway at CaMKII, also mediates the lysosomal degradation of sclerostin protein. Icons outlined red are molecular mechanisms controlling sclerostin abundance that have been described within this manuscript that integrate into our previously described mechano-transduction cascade. Osteoanabolic stimuli, working through reactive oxygen (ROS) and reactive nitrogen species (RNS), direct sclerostin to the lysosome for degradation. This results in reduced sclerostin to allow for bone formation. PM: plasma membrane; ROS: reactive oxygen species; NO: nitric oxide.

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Saline, Labeling, Isolation, Western Blot, Derivative Assay, Control, Recombinant, Transduction, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit Polyclonal Anti-Total CaMKII , Cell Signalling Technology , 3362S RRID: AB_2067938 , (1:1000).

Techniques: Transfection, Construct, Sequencing, In Vitro, In Vivo, Software, Staining

( A ) M281/282 oxidized CaMKII (o-CaMKII) immunoblot showing concentration dependent CaMKIIδ methionine oxidation by ONOO − . Numbers are micromolar concentrations. Ca 2+ and CaM were present. B. CaMKIIδ M281/282 oxidation by 200 μM ONOO − (but not 200 μM dONOO − ) in the presence and absence of Ca 2+ and CaM. t-CaMKII, total CaMKII.

Journal: Scientific Reports

Article Title: Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling

doi: 10.1038/srep23416

Figure Lengend Snippet: ( A ) M281/282 oxidized CaMKII (o-CaMKII) immunoblot showing concentration dependent CaMKIIδ methionine oxidation by ONOO − . Numbers are micromolar concentrations. Ca 2+ and CaM were present. B. CaMKIIδ M281/282 oxidation by 200 μM ONOO − (but not 200 μM dONOO − ) in the presence and absence of Ca 2+ and CaM. t-CaMKII, total CaMKII.

Article Snippet: Membranes were incubated overnight at 4 °C with mouse monoclonal anti-T286 phospho-CaMKII antibody (1:500) (Santa Cruz Biotechnology) and rabbit polyclonal anti-total CaMKII (pan) (1:1000) (Cell Signaling) or with rabbit polyclonal anti-oxidized M281/282 CaMKII antibody (1:2000) (Millipore) and mouse monoclonal anti-total CaMKII antibody (1:1000) (Abcam).

Techniques: Western Blot, Concentration Assay

( A ) Percentage of autonomous activity of CaMKIIδ in the presence and absence of 200 μM ONOO − . The percentage was calculated from activity of apoenzyme in the presence of EGTA in comparison to maximal activity in the presence of Ca 2+ /CaM. One-way ANOVA using Dunnett’s test in comparison to control (No ONOO − ); ***p < 0.001 (n = 5). ( B ) Kinase activity of CaMKIIδ in the presence of Ca 2+ /CaM (Ca 2+ /CaM-dependent activity) and EGTA (Ca 2+ /CaM independent activity) with increasing concentrations of ONOO − and dONOO − . Activity is represented as specific activity, in μmol min −1 mg −1 . Data represent mean values ± s.e.m from 3 experiments. Note the differences in scale for the two y-axes. One-way ANOVA using Dunnett’s post-test compared with control (No ONOO − ); **p < 0.01; ***p < 0.001.

Journal: Scientific Reports

Article Title: Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling

doi: 10.1038/srep23416

Figure Lengend Snippet: ( A ) Percentage of autonomous activity of CaMKIIδ in the presence and absence of 200 μM ONOO − . The percentage was calculated from activity of apoenzyme in the presence of EGTA in comparison to maximal activity in the presence of Ca 2+ /CaM. One-way ANOVA using Dunnett’s test in comparison to control (No ONOO − ); ***p < 0.001 (n = 5). ( B ) Kinase activity of CaMKIIδ in the presence of Ca 2+ /CaM (Ca 2+ /CaM-dependent activity) and EGTA (Ca 2+ /CaM independent activity) with increasing concentrations of ONOO − and dONOO − . Activity is represented as specific activity, in μmol min −1 mg −1 . Data represent mean values ± s.e.m from 3 experiments. Note the differences in scale for the two y-axes. One-way ANOVA using Dunnett’s post-test compared with control (No ONOO − ); **p < 0.01; ***p < 0.001.

Techniques: Activity Assay, Comparison, Control

( A ) Ang II-induced M281/282 oxidation of CaMKIIδ is inhibited by L-NAME, but not by D-NAME. Data are mean values ± s.e.m. from at least 5 experiments. ( B ) Ang II-induced methionine oxidation of CaMKIIδ is inhibited by UA. Data are mean values ± s.e.m. from at least 5 experiments. ***p < 0.001; ****p < 0.0001.

Journal: Scientific Reports

Article Title: Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling

doi: 10.1038/srep23416

Figure Lengend Snippet: ( A ) Ang II-induced M281/282 oxidation of CaMKIIδ is inhibited by L-NAME, but not by D-NAME. Data are mean values ± s.e.m. from at least 5 experiments. ( B ) Ang II-induced methionine oxidation of CaMKIIδ is inhibited by UA. Data are mean values ± s.e.m. from at least 5 experiments. ***p < 0.001; ****p < 0.0001.

Techniques:

( A ) Ang II-induced T287 autophosphorylation of CaMKIIδ (p-CaMKII) is inhibited by L-NAME, but not D-NAME. Data are mean values ± s.e.m. from at least 7 experiments. ( B ) Ang II-induced T287 CaMKIIδ autophosphorylation is inhibited by UA. Data are mean values ± s.e.m. from at least 6 experiments. *p < 0.05; ****p < 0.0001.

Journal: Scientific Reports

Article Title: Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling

doi: 10.1038/srep23416

Figure Lengend Snippet: ( A ) Ang II-induced T287 autophosphorylation of CaMKIIδ (p-CaMKII) is inhibited by L-NAME, but not D-NAME. Data are mean values ± s.e.m. from at least 7 experiments. ( B ) Ang II-induced T287 CaMKIIδ autophosphorylation is inhibited by UA. Data are mean values ± s.e.m. from at least 6 experiments. *p < 0.05; ****p < 0.0001.

Techniques:

Review

Structured Review

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