rabbit polyclonal anti camkii pan  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt , Mfn2 -null MEFs with vec , CaMKII-WT, or CaMKII-DN in the μ-slide. Time-lapse images were taken every 10 min for 17 hr and 50 min. MEFs were tracked for velocity quantification. Scale bar: 50 μm.

Article Snippet: Antibody , Anti-pan-CaMKII (rabbit polyclonal) , Cell Signaling Technology , #3362 , WB (1:1000).

Techniques:

Journal: iScience

Article Title: Exercise-acclimated microbiota improves skeletal muscle metabolism via circulating bile acid deconjugation

doi: 10.1016/j.isci.2023.106251

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CaMKII , Cell Signaling Technology , Cat#4436S; RRID: AB_10545451.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Gene Expression, Software, Mass Spectrometry, Real-time Polymerase Chain Reaction

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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis

doi: 10.1155/2021/6924251

Figure Lengend Snippet: Primary and secondary antibodies used for Western blotting.

Article Snippet: Anti-CaMKII (pan) rabbit polyclonal antibody , 1 : 500 in Roti-TBS-T (#1061.1, Roth, Germany) , #3362, Cell Signaling, USA.

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis

doi: 10.1155/2021/6924251

Figure Lengend Snippet: Abnormal fission-related proteins in cervical wobbler spinal cord at p40. (a) mRNA expression levels of Mfn1 , Mfn2 , Opa1 , Oma1 , and Dnm1l from the stable phase of wild-type (WT) and wobbler (WR) spinal cords were investigated by qPCR. mRNA levels were significantly reduced in WR except Dnm1l . For relative quantification, the 2 −∆∆Ct method was conducted using GAPDH for normalization. N = 7-11 per genotype. (b) Exemplary Western blots of Mfn1 (≈85 kDa), Mfn2 (≈85 kDa), Opa1 (≈100 kDa), and Oma1 (≈50 kDa) in the cervical spinal cord of p40 WT and WR. Actin (≈45 kDa) and calnexin (≈90 kDa) were used as control proteins. Bar charts represent the semiquantitative analysis of protein expression levels. Western blots revealed unchanged expression of Mfn1, Opa1, and Oma1 as well as significantly increased expression of Mfn2 in the cervical spinal cord of wobbler mice. N = 8-10 per genotype. (c) Exemplary Western blots of Drp1 (≈85 kDa), p-Drp1 (Ser616; ≈85 kDa), and actin (≈45 kDa) as control protein. Analysis of band intensity is presented in bar charts. Total amount of Drp1 does not differ between the two genotypes; Drp1 is significant more often phosphorylated at Ser616 in cervical spinal cords of wobbler mice. N = 9 per genotype. (d) Exemplary Western blots of CaMKII (≈55 kDa) and oxidized Ox-CaMKII (Met281/282; ≈55 kDa) in combination with calnexin (≈90 kDa) as control protein from wild-type and wobbler cervical spinal cords. Analysis of band intensity is presented in bar charts and showed equal CaMKII levels; thus, the proportion of oxidized CaMKII at Met281/282 is significantly increased in wobbler spinal cords compared to wild-type. N = 7 per genotype. All data are presented as the mean values ± SEM, and Student's t -test was performed for significance testing between WT and WR. Values with p < 0.05 were considered to be significant. Significant differences are indicated by ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-CaMKII (pan) rabbit polyclonal antibody , 1 : 500 in Roti-TBS-T (#1061.1, Roth, Germany) , #3362, Cell Signaling, USA.

Techniques: Expressing, Quantitative Proteomics, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis

doi: 10.1155/2021/6924251

Figure Lengend Snippet: Proposed mechanism of motor neuronal cell death in wobbler mice. An impaired function of complexes I and III of the mitochondrial respiratory chain leads to an increase in superoxide anions. Increased superoxide anion levels cause an oxidation and thus calcium-independent activation of CaMKII. Ox-CaMKII in turn stimulates phosphorylation of Drp1 at Ser616, which recruits it to the mitochondrial membrane and causes enhanced mitochondrial fission. This disruption between fusion and fission balance promotes fragmentation of the mitochondrial network, resulting in increased production of reactive oxygen species. This is likely to trigger a nonreversible process that leads to fragmented and dysfunctional mitochondria, resulting in a self-reinforcing vicious circle that promotes degeneration of motor neurons in wobbler mice.

Article Snippet: Anti-CaMKII (pan) rabbit polyclonal antibody , 1 : 500 in Roti-TBS-T (#1061.1, Roth, Germany) , #3362, Cell Signaling, USA.

Techniques: Activation Assay, Phospho-proteomics, Membrane, Disruption