Journal: International journal of molecular sciences

Article Title: Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

doi: 10.3390/ijms24054718

Figure Lengend Snippet: Figure 4. Calpastatin is enhanced in CaMKK2 deficient osteocyte conditioned media from female mice. (A) Scheme for primary osteocyte isolation from the long bones of individual female and male control and Camkk2∆OCY mice and collection of serum-free conditioned media for LC-MS/MS analysis. (B) Principal component analysis (PCA) plot of proteomic signatures of osteocyte CM (n = 3/group). (C) Venn diagram of proteins with increased or decreased abundance in female and male Camkk2∆OCY osteocyte CM relative to sex-matched controls. (D) Pie chart showing functional annotation of osteocyte CM proteins. All GO-Terms with a minimum False Discovery Rate (FDR) corrected p-value < 0.05 were included. (E,F) Volcano plots of enriched proteins in female and male Camkk2∆OCY osteocyte CM relative to sex-matched controls. Calpastatin (Cast) is represented within the

Article Snippet: Quantification of calpastatin levels in osteocyte CM was performed using the Mouse CAST (Calpastatin) ELISA kit (NBP2-75048; Novus Biologicals, Centennial, CO, USA) according to manufacturer’s directions.

Techniques: Isolation, Control, Liquid Chromatography with Mass Spectroscopy, Functional Assay

Journal: International journal of molecular sciences

Article Title: Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

doi: 10.3390/ijms24054718

Figure Lengend Snippet: Figure 5. Depletion of Calpastatin from conditioned media of female CaMKK2-deficient osteocytes restores osteoclast function. (A) Schematic of experimental design outlining the collection of female osteocyte CM and depletion of Cast by immunoprecipitation (IP) with α-Cast antibody or control

Article Snippet: Quantification of calpastatin levels in osteocyte CM was performed using the Mouse CAST (Calpastatin) ELISA kit (NBP2-75048; Novus Biologicals, Centennial, CO, USA) according to manufacturer’s directions.

Techniques: Immunoprecipitation, Control

Journal: PLOS ONE

Article Title: Expression of RNA polymerase I catalytic core is influenced by RPA12

doi: 10.1371/journal.pone.0285660

Figure Lengend Snippet: (A) qPCR analysis of RPA12 transcript in A375 melanoma cells with transient RPA12 knockdown using siRNA. Mean fold change ± SD of n = 4 biological replicates is shown. Mann-Whitney two-tailed t-test *, p < 0.05. (B) Immunofluorescence staining of RPA12. Representative biological replicate of n = 4 is shown. (C) Quantification of the images for RPA12. Mean fold change ±SD of n = 3 biological replicates is shown. Analysis of N = 50 cells per treatment. Student’s two-tailed t-test **, p = 0.0018. (D) Immunofluorescence staining of siCtrl and siRPA12 knockdown cells. A375 melanoma cells were stained for the indicated Pol I subunits and fibrillarin (FBL) following siRNA knockdown of RPA12. DNA was counterstained using Hoechst. Representative biological replicates of n = 4 are shown. (E) Quantification of the images for RPA194. Mean fold change ±SD of n = 3 biological replicates is shown. N = 50–100 cells per treatment. Student’s two-tailed t-test *, p = 0.0306. (F) Immunofluorescence staining of siCtrl and siRPA12 knockdown cells for PAF53 and CAST. (G) Quantification of the images for PAF53 and CAST. N = over 100 cells per treatment. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: RPA135 (mouse monoclonal 4H6 1:100; sc-293272 Santa Cruz Biotechnology), RPA194 (mouse monoclonal C-1 1:100; sc-48385 Santa Cruz Biotechnology), PAF53 (rabbit polyclonal 16145-1-AP 1:200; ProteinTech), CAST (rabbit polyclonal PA5-27632 1:200; Bethyl Laboratories), ZNRD1 (RPA12) (mouse monoclonal D10 1:100; sc-393406 Santa Cruz Biotechnology) and fibrillarin (rabbit polyclonal 1:500; ab5821 Abcam).

Techniques: Knockdown, MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs

doi: 10.3390/ijms23031391

Figure Lengend Snippet: The treatment of n-BP rescued the progression of QA-induced excitotoxicity in SCA3 PPs. The control and/or SCA3 PPs were treated with or without QA (1 μM) in the presence of n-BP (0, 10, 20, and 40 μg/mL) for 12 h. ( A ) Images of QA-treated SCA3 PPs in the presence of BP (40 μg/mL). ( B ) Confocal images of poly Q localization in BP (40 μg/mL)-treated SCA3 PPs in the presence of QA (1 um). When compared with C, 40 μg/mL of n-BP prevented the amounts of colocalized polyQ in the cell nucleus of PPs. The distribution of polyQ in the nucleus is indicated by white arrowheads. Scale bar = 100 μm; ( C ) protein analysis of wild-type, mutant ATXN3 and its proteolytic fragment in cell lysates of control and SCA3 PPs, as assessed by immunoblots. Soluble, soluble protein; Insoluble, insoluble protein; ( D , E ) representative immunoblot of control and cleaved PARP1 in cell lysates of control and SCA3 PPs, as assessed by immunoblots. The HDAC2 were used as loading controls. The quantification from three independent images is presented as the means ± standard deviation. t(4) = 2.824, p < 0.05, for lane 1 vs. lane 2; t(4) = 5.613, p < 0.01, for lane 1 vs. lane 3; t(4) = 3.522, p < 0.05, for lane 1 vs. lane 4; ( F ) the quantification result of calcium concentration in SCA3 PPs, as assessed by Fura-2 indicator. Cells were treated with QA (1 μM) in the presence of n-BP (0, 10, 20, and 40 μg/mL). Data are presented as the means ± standard deviation. t(4) = 2.95, p < 0.05; ( G ) the calpain activity in control and SCA3 PPs cell lysates, as assessed by ELISA assay. The quantification results from three independent replicates are presented as the means ± standard deviation. t(4) = 3.27, p < 0.05, for lane 3 vs. lane 4; t(4) = 17.55, p < 0.01, for lane 3 vs. lane 5; t(4) = 9.019, p < 0.01, for lane 3 vs. lane 6. ( H ) Protein analysis of calpain 1, calpain 2 and calpastatin in cell lysates of SCA3 PPs, as assessed by immunoblots. Cells were treated with or without QA (1 μM) in the presence of n-BP (0, 10, 20, and 40 μg/mL). *, p < 0.05; **, p < 0.01.

Article Snippet: The primary antibodies used in this study were as follows: ATXN3 (1:1000; MAB5360, Millipore), calpain 1 (1:5000; GTX102340, GeneTex, Irvine, CA, USA), calpain 2 (1:5000; GTX111809, GeneTex), calpastatin (1:5000; GTX645540, GeneTex), PARP1 (1:1000, MAB600, R&D), GAPDH (1:10,000, ab8245, Abcam), β-actin (1:5000; MAB8929, R&D), and HDAC2 (1:5000; GTX109642, GeneTex).

Techniques: Control, Mutagenesis, Western Blot, Standard Deviation, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Life

Article Title: Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

doi: 10.3390/life13030854

Figure Lengend Snippet: Phosphorylation images of calpastatin in the three groups after incubation at 30 °C for 30 min. ( a ) Phosphorylation of calpastatin in the three groups after incubation at 30 °C for 30 min; ( b ) relative phosphorylation levels of calpastatin in the three groups. a–c represents significant difference between groups ( p < 0.05).

Article Snippet: μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem (San Diego, CA, USA) and Merck-Millipore (Burlington, MA, USA), respectively.

Techniques: Phospho-proteomics, Incubation

Journal: Life

Article Title: Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

doi: 10.3390/life13030854

Figure Lengend Snippet: Phosphorylation levels of calpastatin in three groups with different ratios between calpastatin and calpain during incubation at 4 °C. Calpastatin:calpain = 1:2.5 ( a ); 1:5 ( b ); 1:10 ( c ); 1:15 ( d ). a–b represents significant difference between groups ( p < 0.05).

Article Snippet: μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem (San Diego, CA, USA) and Merck-Millipore (Burlington, MA, USA), respectively.

Techniques: Phospho-proteomics, Incubation

Journal: Life

Article Title: Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

doi: 10.3390/life13030854

Figure Lengend Snippet: Relative degradation rate of μ-calpain in three groups with different ratios between calpastatin and calpain during incubation at 4 °C. Calpastatin:calpain = 1:2.5 ( a ); 1:5 ( b ); 1:10 ( c ); 1:15 ( d ). a–c represents significant difference between groups ( p < 0.05).

Article Snippet: μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem (San Diego, CA, USA) and Merck-Millipore (Burlington, MA, USA), respectively.

Techniques: Incubation

Journal: Life

Article Title: Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

doi: 10.3390/life13030854

Figure Lengend Snippet: Degradation of calpastatin in three groups with different ratios between calpastatin and calpain during incubation at 4 °C. Calpastatin:calpain = 1:2.5 ( a ); 1:5 ( b ); 1:10 ( c ); 1:15 ( d ).

Article Snippet: μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem (San Diego, CA, USA) and Merck-Millipore (Burlington, MA, USA), respectively.

Techniques: Incubation

Journal: Life

Article Title: Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

doi: 10.3390/life13030854

Figure Lengend Snippet: Identification of phosphorylated peptides and phosphorylated sites of calpastatin in the three groups with different phosphorylation levels.

Article Snippet: μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem (San Diego, CA, USA) and Merck-Millipore (Burlington, MA, USA), respectively.

Techniques: Phospho-proteomics, Control

Journal: Life

Article Title: Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

doi: 10.3390/life13030854

Figure Lengend Snippet: A 3D simulated diagram of calpastatin with two phosphorylation sits was obtained from the Uniprot database ( http://www.uniprot.org/ , accession number: P20810, accessed on 25 January 2023). The left side of the figure shows the specific sites in the calpastatin where calpastatin is phosphorylated.

Article Snippet: μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem (San Diego, CA, USA) and Merck-Millipore (Burlington, MA, USA), respectively.

Techniques: Phospho-proteomics