Journal: EMBO Reports

Article Title: A CRISPR-Cas9 screen reveals genetic determinants of the cellular response to decitabine

doi: 10.1038/s44319-025-00385-w

Figure Lengend Snippet: ( A ) Dose–response curves of HAP1 cells transduced with sgCTRL or two different sgRNAs targeting KDM1A (sg KDM1A #1 and #2) upon DAC treatment at indicated concentrations. Cell viability was measured by CellTiter-Glo after 3 days of DAC treatment. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. ( B ) Dose–response curves of wild-type HAP1 cell and isogenic KDM1A KO clones upon DAC treatment at indicated concentrations. Cell viability was measured by CellTiter-Glo after 3 days of DAC treatment. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. ( C ) Survival analysis of WT, KDM1A KO clones, and KDM1A KO clones complemented with either empty vector, wild-type KDM1A (KDM1A-WT), or catalytically inactive KDM1A-AE/KA and KDM1A-3DA mutants after treatment with DAC at indicated concentrations for 3 days. Cell viability was measured by CellTiter-Glo. Data represent the means ± SEM of 2 biological independent clones (2 technique replicates each, n = 4). The p values were determined using two-way ANOVA followed by Dunnett’s multiple comparisons test. ( D ) Immunoblot analysis showing the KDM1A protein level in two HAP1 clones expressing FKBP-KDM1A fusion protein treated with or without dTAG-13 for 2 days with β-ACTIN as a loading control. ( E ) Dose–response curves of two HAP1 clones expressing FKBP-KDM1A fusion protein upon DAC treatment at indicated concentration. Cells were treated with or without dTAG-13 at the same time with DAC. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. ( F ) Heatmap and bar plot (left) depicting sgRNA abundance changes of KDM1A domain scanning at indicated time points versus Day 0, normalized against non-targeting control sgRNAs. The positions of domain scanning sgRNAs in the coding region of KDM1A are indicated as vertical lines in the above schematic diagram. Heatmap (right) for averaged differential β scores of sgRNAs in KDM1A domains with clustering. ( G ) Survival analysis of WT, and KDM1A KO clones, and KDM1A KO clones complemented with KDM1A-ΔNFR, KDM1A-ΔSWIRM, KDM1A-ΔNFR/ΔSWIRM, and KDM1A-ΔTOWER after treatment with DAC at indicated concentrations for 3 days. Cell viability was measured by CellTiter-Glo. Data represent the means ± SEM of 2 biological independent clones (2 technical replicates each, n = 4). The p values were determined using two-way ANOVA followed by Dunnett’s multiple comparisons test. ( H ) KDM1A ChIP-seq metagene profiles of KDM1A binding peaks in KDM1A KO clone complemented with KDM1A-WT, KDM1A-ΔSWIRM, and KDM1A-ΔTOWER (upper). Heatmap representation of KDM1A occupancies at KDM1A binding peaks in these cells ranked by the intensity of KDM1A ChIP-seq signal (lower). ( I ) Volcano plot showing differential interacted proteins between KDM1A-WT and KDM1A-ΔSWIRM obtained from label-free quantification MS with triplicates. The p values were determined by two-sample t -test using a permutation-based FDR approach. ( J ) Immunoblot analysis for immunoprecipitations of 3×FLAG-tagged KDM1A-WT and KDM1A-ΔSWIRM. ( K ) Dose–response curves of wild-type HAP1 and isogenic ZMYM2 (left) or ZMYM3 (right) KO clones upon DAC treatment at indicated concentrations. Cell viability was measured by CellTiter-Glo after 3 days of DAC treatment. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. .

Article Snippet: FKBP12 PROTAC dTAG-13 , MCE , HY-114421.

Techniques: Transduction, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Control, Concentration Assay, ChIP-sequencing, Binding Assay, Quantitative Proteomics

Journal: EMBO Reports

Article Title: A CRISPR-Cas9 screen reveals genetic determinants of the cellular response to decitabine

doi: 10.1038/s44319-025-00385-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: FKBP12 PROTAC dTAG-13 , MCE , HY-114421.

Techniques: Recombinant, Sequencing, Software, Cloning, Proliferation Assay, In Situ

Journal: The Journal of Biological Chemistry

Article Title: FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury

doi: 10.1016/j.jbc.2022.101647

Figure Lengend Snippet: FKBPL and FKBP8 induced lysosome-dependent DLK degradation. A , comparative analysis of relative expression levels in mouse L4,5 dorsal root ganglion (DRG) tissues, sciatic nerve tissues, and cultured embryonic DRG neurons ( , , , ). Red and blue circles indicate Illumina short-read sequencing and Oxford Nanopore direct RNA long-read sequencing, respectively. Circle sizes indicate relative levels of microarray data from cultured embryonic DRG neurons. B , Western blot analysis for the expression of DLK with FKBPs (N; null vector, L; FKBPL, 3; FKBP3, 4; FKBP4, 8; FKBP8, 12; FKBP12, 14; FKBP14, 15; FKBP15). The number indicates normalized relative intensity. Dual leucine zipper kinase and FLAG-epitope-tagged FKBP protein family members were expressed in HEK293T cells and subjected to SDS-PAGE. C , Western blot analysis for the immunoprecipitation of DLK with mouse (m) and human (h) FKBP4/8 that was overexpressed in HEK293T cells. Empty arrowhead , non-specific band; blue arrowhead , FKBP4; red arrowhead , FKBP8. D , Western blot analysis for the expression of DLK and FKBPL/4/8 expressed in HEK293T cells with or without bafilomycin A1 treatment. The numbers indicate the normalized relative intensity. E , Western blot analysis of DLK protein levels under Fkbp8 knockdown (sh Fkbp8 ) by lentiviral delivery in primary cultured embryonic DRG neurons. The numbers indicate the normalized relative intensity. F , statistical analysis of ( E ) (FC, fold change; n = 3 for each condition; ∗ p < 0.05 by t test; mean ± S.E.M.). DLK, dual leucine zipper kinase.

Article Snippet: An expression plasmid for full-length Myc-DDK-tagged Mouse Fkbp3 (MR202616), Fkbp4 (MR227193), Fkbp8 (MR220865), Fkbp12 (MR200405), Fkbp14 (MR202290), and Fkbp15 (MR220579) were purchased from Origene.

Techniques: Expressing, Cell Culture, Sequencing, Microarray, Western Blot, Plasmid Preparation, FLAG-tag, SDS Page, Immunoprecipitation, Knockdown

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 1 Expression of FKBP12 and CyPA in several organs and tissues. mRNA expression of FK506 binding protein 12 (FKBP12) and cyclophilin A (CyPA) was detected in several major organs, including cerebrum, thymus, spleen, lung, heart, liver, and kidney. mRNA expression of FKBP12 in lymphoid tissues, thymus and spleen, was lower than that of CyPA. FKBP12 and CyPA mRNA were abundantly expressed in the lung, heart, and liver. In the kidney, mRNA expression of CyPA was higher than that of FKBP12. Although CyPA mRNA was equally expressed in all fractions of kidney samples, the expression of FKBP12 mRNA in glomeruli was higher than that in cortex and medulla. Representative RT-PCR findings were shown in upper panel. The band intensity was semi-quantified by the densitometry. The quantitative analysis of the distribution of each fraction in the kidney was shown in lower panel. The data are expressed as a percentage of each fraction in the total of three fractions (glomeruli, cortex, and medulla) (means ± SD, n = 3; ***p < .005, t-test)

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 2 Localization of FKBP12 and CyPA in glomeruli. (A) Dual-labeling immunofluorescence (IF) findings of FKBP12 (green) with glomerular cell markers (red) in adult rat glomeruli. The staining of FKBP12 was clearly apart from that of RECA-1 and Thy1.1, indicating that FKBP12 is not expressed in endothelial cells or mesangial cells, and FKBP12 is restricted in podocytes. In podocytes, major portions of the FKBP12 staining were co-stained with synaptopodin, a foot process marker. A part of FKBP12 staining was co-localized with nephrin, podocalyxin, and integrin-α3 (arrows). (B) Dual-labeling IF findings of CyPA (green) with glomerular cell markers (red). The staining of CyPA was apart from that of RECA-1. A part of the CyPA staining was co-localized with Thy1.1. Some portions of the CyPA were co-localized with synaptopodin, nephrin, podocalyxin, and integrin-α3 (arrows). (C) Dual-labeling IF findings of FKBP12 (green) with nephrin (red) in neonatal rat glomeruli. FKBP12 staining was clearly detected in a maturing podocyte, and the staining was restrictedly detected at the basal side of the podocyte as a continuous fine granular pattern slightly before nephrin appeared (arrowhead). The staining of FKBP12 was co-localized with that of nephrin in the capillary loop stage. Scale bar, 20 μm

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Labeling, Immunofluorescence, Staining, Marker

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 3 FKBP12 is co-localized with 14-3-3β and associates with F-actin in podocytes. (A) mRNA expression of FKBP12 and CyPA in undifferentiated and differentiated human cultured podocytes. Although no difference in mRNA expression of CyPA was detected between the undifferentiated podocytes and the differentiated podocytes, FKBP12 mRNA expression in the differentiated cultured podocytes was higher than that in the undifferentiated cells. The semiquantitative data of the expressions of FKBP12 and CyPA corrected by that of GAPDH are expressed as means ± SD (n = 4; ***p < .005, t-test). (B) Western blot analysis of sequentially solubilized cell lysates of the cultured podocytes. Clear bands of FKBP12 were observed in F1 (Triton X-100-soluble fraction) and F2 (Triton X-100-insoluble/RIPA-soluble fraction). The band of FKBP12 in F2 was higher than that in F1. Bands of CyPA were equally detected in F1 and F2, and a weak one was observed in F3 (RIPA-insoluble fraction). Clear bands of 14-3-3 were detected in F1 and F2. The band of 14-3-3 in F2 was higher than that in F1. The distribution of each fraction in total factions was evaluated by the densitometric analyses. The data are expressed as a percentage of each fraction in total fractions (F1, F2, and F3) (means ± SD, n = 5; ***p < .005, t-test). (C) Immunostaining of FKBP12 and CyPA in human cultured podocytes. Clear fibrous staining of FKBP12 toward the tip end of processes was detected in the cytoplasm in podocytes. Although weak fibrous staining of CyPA was detected, CyPA staining was broadly detected in the cytoplasm of podocytes. Scale bar, 20 μm. (D) Dual immunostaining of FKBP12 and F-actin in cultured podocytes. The fibrous staining of FKBP12 was co-localized with F-actin in the processes of podocytes. Scale bar, 20 and 10 μm. (E) Actin binding assay with the cell lysate of cultured podocytes. The specific bands of FKBP12 and 14-3-3 were detected in the sample incubated with F-actin. No band was detected in the sample incubated with monomeric actin (mono- Actin). (F) Dual immunostaining of FKBP12 and 14-3-3β in glomeruli. FKBP12 was co-localized with 14-3-3β in glomeruli. Scale bar, 20 μm

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Expressing, Cell Culture, Western Blot, Immunostaining, Staining, Binding Assay, Incubation

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 4 FKBP12 interacts with the actin-associated proteins 14-3-3β and synaptopodin. (A) The immunoprecipitation (IP) assays with the HEK293 cells transfected with FKBP12-RFP. The band of endogenous 14-3-3 was detected in the precipitate with anti-RFP antibody (upper panel). FKBP12 band was detected in the precipitate with anti-pan14-3-3 antibody (lower panel). (B) The assays with the cells co-transfected with FKBP12-RFP and synaptopodin (Synp)-HA. The band of Synp was detected in the precipitate with anti-RFP antibody (upper panel). FKBP12 band was detected in the precipitate with anti-HA antibody (lower panel). (C) The assays with the cells transfected with Synp-HA. The band of Synp was detected in the precipitate with anti-pan14-3-3 antibody. (D) The assay with the cultured podocytes. The band of FKPB12 was detected in the precipitate with anti-14-3-3β. (E) The assay of the interaction of FKBP12 with synaptopodin in HEK cells treated with 14-3-3β siRNA. The band intensity of synaptopodin was corrected by that of FKBP12. The data are shown as a ratio relative to the control siRNA-treated cells. The data of three independent experiments are expressed as means ± SD. The amount of Synp in the FKBP12 precipitate was not altered by the deletion of 14- 3-3β (upper panel). The band intensity of FKBP12 was corrected by that of synaptopodin. The amount of FKBP12 in the precipitate of synaptopodin was not altered by the 14-3-3β deletion (lower panel) (n = 3; t-test). (F) The assays of the interaction of 14-3-3β with FKBP12 in the cells transfected with synaptopodin (upper panel). No difference in the amount of endogenous 14-3-3β in the precipitate of FKBP12 was detected between the cells transfected with synaptopodin and the cells not transfected. The assays of the interaction of 14-3-3b with synaptopodin in the cells transfected with FKBP12 (lower panel). No difference in the amount of endogenous 14-3-3β in the precipitate of synaptopodin was detected between the cells transfected with FKBP12 and the cells not transfected (means ± SD, n = 3; t-test). NMS, normal mouse serum; NRS, normal rabbit serum

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Immunoprecipitation, Transfection, Cell Culture, Control

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 5 Deranged F-actin structure, impaired process formation, and decreased expression of 14-3-3β are detected in FKBP12 knockdown podocytes. (A) F-actin staining in the cultured podocytes treated with FKBP12 siRNA for 48 h. F-actin staining was clearly decreased in the cells treated with FKBP12 siRNA. The proportion of the cells forming processes (arrow) was decreased in FKBP12 knockdown podocytes (n = 4; *p < .05, U test, n = 3; ***p < .005, t-test). Scale bar, 20 μm. (B) Expression and phosphorylation of 14-3-3β in the cultured podocytes treated with FKBP12 siRNA for 72 h. Although the alteration in the band intensity of phosphorylated 14-3-3 was not detected, the expression of total 14-3-3β was decreased in the cultured podocyte treated with FKBP12 siRNA (63.0% ± 7.6%). The band intensity of FKBP12 was decreased in the cells treated with FKBP12 siRNA (36.2% ± 5.8%). The data are shown as a percentage relative to the control siRNA-treated cells and are expressed as means ± SD (n = 3; *p < .05, **p < .01, t-test)

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Expressing, Knockdown, Staining, Cell Culture, Phospho-proteomics, Control

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 7 Tacrolimus (Tac) treatment suppresses the decrease of FKBP12 in adriamycin-induced podocyte injury. (A) Dual-labeling immunofluorescence (IF) findings of podocin (green) and nephrin (red) in adriamycin (ADR) nephropathy treated with Tac. The decrease of nephrin and podocin staining in glomeruli was suppressed by Tac treatment on day 28 of ADR nephropathy. The score is shown as means ± SD (n = 4; *p < .05, U test). Scale bar, 20 μm. (B) IF staining of FKBP12 in ADR nephropathy treated with Tac. Tac treatment suppressed the decrease of FKBP12 staining in ADR nephropathy (n = 4; *p < .05, U test). Scale bar, 20 μm. (C) F-actin staining and expression of nephrin mRNA in the injured podocyte treated with Tac. Tac treatment suppressed the decrease of F-actin staining in the cultured podocyte treated with ADR (n = 4; *p < .05, U test). The proportion of the cells forming the processes to total cells was decreased in the podocytes treated with ADR. Tac treatment suppressed the decrease in the injured podocytes (arrow) (n = 3; *p < .05, t-test). The decrease of nephrin mRNA in the injured podocytes was suppressed by Tac treatment. Scale bar, 20 μm. (D) Western blot findings of FKBP12 in the injured podocyte treated with Tac. The protein expression of FKBP12 was decreased in the injured podocytes. The decrease was suppressed by Tac treatment (n = 3; *p < .05, t-test). (E) Dual-labeling IF findings of FKBP12 (green) with F-actin (red). Tac treatment restored the FKBP12 at F-actin in processes of the podocytes (arrowhead). Scale bar, 20 μm

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Labeling, Immunofluorescence, Staining, Expressing, Cell Culture, Western Blot

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 9 Schematic diagram of the mechanism of how tacrolimus (Tac) ameliorates podocyte injury. (A) In normal podocytes, FKBP12 is expressed along the actin cytoskeleton and associates with F-actin. FKBP12 interacts with 14-3-3 and synaptopodin to maintain F-actin. (B) In injured podocyte, FKBP12 is downregulated, and the downregulation of FKBP12 causes the decrease in the expression of 14-3-3 and the disruption of 14-3-3-synaptopodin linkage. Synaptopodin dissociated from 14-3-3 is easily degraded. Decrease in the expression of FKBP12 and the consequent disruption of the linkages of FKBP12 with 14-3-3 and synaptopodin results in deranged F- actin structure. (C) Tac treatment enhances the interaction of FKBP12 with synaptopodin and suppresses the decrease of FKBP12. Tac treatment suppresses the decrease of process formation in podocyte injury by restoring FKBP12 at actin cytoskeleton

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Expressing, Disruption

Journal: The FASEB Journal

Article Title: Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton

doi: 10.1096/fj.202101052r

Figure Lengend Snippet: FIGURE 8 Tacrolimus (Tac) treatment to normal cultured podocytes increases FKBP12 expression at the tip end of processes and enhances process formation. (A) F-actin staining in the cultured podocytes treated with Tac. The staining intensity of F-actin was not altered in the cells treated with Tac. The treatment increased the proportion of the cells forming processes (arrow). The data are expressed as means ± SD (n = 4; U test, n = 3; ***p < .005, t-test). Scale bar, 20 μm. (B) Dual-labeling immunofluorescence findings of FKBP12 (green) with F-actin (red). The expression of FKBP12 at actin cytoskeleton in the tip end of processes was increased by Tac treatment (arrowhead). Scale bar, 20 μm. (C) Western blot analysis of sequentially solubilized cell lysates of the cultured podocytes treated with Tac. The proportion of FKBP12 in Triton X-100-soluble fraction (F1) to total FKBP12 was increased by Tac treatment. The treatment decreased the proportion of FKBP12 in Triton X-100-insoluble/RIPA soluble fraction (F2) to total FKBP12. As well as FKBP12, the treatment increased the proportion of 14-3-3β in the F1 and decreased the proportion of 14-3-3β in F2 to total 14-3-3β. The alteration of total expression in FKBP12 and 14-3-3β was not detected in the podocytes treated with Tac. The treatment did not alter the proportions of CyPA in F1 and F2. Tac treatment did not alter the expression of total CyPA. The data are expressed as means ± SD (n = 3; *p < .05, t-test). (D) The immunoprecipitation assays with the HEK293 cells co-transfected with FKBP12-RFP and synaptopodin (Synp)-HA. 14-3-3β and FKBP12 in the precipitate with anti-HA antibody were increased by Tac treatment. (E) The effect of Tac on phosphorylation of 14-3-3 and Synp were analyzed with the HEK cells co-transfected with FKBP12-REP and Synp-HA. Transfected Synp-HA was detected as an approximately 130 kDa band. The same size band detected with anti-phospho-serine antibody showed phosphorylated Synp. Phosphorylation levels of 14-3-3 and Synp were not altered by Tac treatment

Article Snippet: Assays with HEK293 cell transfection were performed as previously described.25 HEK293 cells were transfected with monomeric red fluorescent protein (RFP)- FKBP12 (Addgene, Cambridge, MA, USA, Plasmid #67514) and synaptopodin- HA by the calcium phosphate method.

Techniques: Cell Culture, Expressing, Staining, Labeling, Immunofluorescence, Western Blot, Immunoprecipitation, Transfection, Phospho-proteomics

Journal: CNS Neuroscience & Therapeutics

Article Title: DNLA Delayed the Appearance of Learning and Memory Impairment of APP / PS1 Mice: Involvement of mTOR / TFEB /v‐ ATPase Signaling Pathway

doi: 10.1111/cns.70300

Figure Lengend Snippet: The effect of DDB on mTOR. (A) Co‐localization of mTOR and lysosome (scale bar = 20 μm). ( x ¯ ± SD, n = 4). (B and C) Representative bands and quantitative statistics of protein expression levels for p‐mTOR (ser2448), p‐S6K (pT389) are shown. ( x ¯ ± SD, n = 5). (D and E) DARTS samples with WB for FKBP12 and mTOR protein quantified relative to its Control. ( x ¯ ± SD, n = 5), * p < 0.05, ** p < 0.01 vs. Control group.

Article Snippet: FKBP12 (#55104) was obtained from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: [ 3 H]S107 binding to SR vesicles.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A) Representative immunoblot of SR vesicles not treated and treated with FK506. SR vesicles were incubated with 10 µM FK506 as described in , followed by centrifugation to remove FK506 and dissociated FKBP12. (B and C) FKBP12 dissociation from SR vesicles in the presence of GSH and GSSG. Immunoblots of SR vesicles not treated with FK506 were incubated for 1 and 20 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 50 µM free Ca 2+ , protease inhibitors, and the indicated concentrations and ratios (5 mM total glutathione) of GSH and GSSG in the absence and presence of 44 µM S107. Free FKBP12 was removed by centrifugation. Data were normalized to SR vesicles not incubated (gray bar, 0 min) and are the mean ± SEM of 4–5 experiments. *p<0.05 compared to SR vesicles at 0 min not treated with S107.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Western Blot, Incubation, Centrifugation

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A–C) SR vesicles not treated with FK506 were incubated for 5 h at 24°C with or without 0.10 mM NOC12 in the absence and presence of 44 µM S107 in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca 2+ and protease inhibitors. S-nitrosylation was stopped by centrifugation. Resuspended samples were separated on 8–20% (FKBP12) and 3–12% (RyR1 and Cys-SNO) gradient SDS-PAGE gels and transferred to nitrocellulose membranes to detect S-nitrosylation of RyR1, and FKBP12 and RyR1 proteins. Data are the mean ± SEM of 4 determinations. *p<0.05 compared to control samples (B) and samples with NOC12 and S107 (C). (D and E) SR membranes were incubated with and without 44 µM S107 and 0.1 mM NOC12 at 24°C for 90 min, solubilized, and immunoprecipitated as described in Methods. Immunoblots of RyR1 and FKBP12 are shown. Data are the mean ± SEM of 4 experiments. * p<0.05 compared to control samples and samples incubated with NOC12 and S107.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Incubation, Centrifugation, SDS Page, Control, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A) Dependence of [ 3 H]ryanodine binding on NOC12 concentration. SR vesicles not treated with FK506 were incubated for 5 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca 2+ , protease inhibitors and the indicated concentrations of NOC12 in the presence (•) and absence (○) of 44 µM S107. Data are the mean ± SEM of 4 experiments. *p<0.05 compared to vesicles without S107. (B) Dependence of [ 3 H]ryanodine binding to RyR1 on S107 concentration. SR vesicles were incubated as in A in the absence (○) and presence of 50 µM NOC12 (•) and the indicated concentrations of S107. Data are the mean ± SEM of 4 experiments. *p<0.05 compared to vesicles with 50 µM NOC12 and without S107. (C) Specific [ 3 H]ryanodine binding to SR vesicles containing (−FK506) and depleted (+FK506) of FKBP12. [ 3 H]Ryanodine binding was determined in the presence of 50 µM NOC12 and the absence and presence of 44 µM S107. Data are the mean ± SEM of 8 experiments. *p<0.05 compared to vesicles treated with FK506 and incubated in the absence of S107, # p<0.05 compared to vesicles not treated with FK506 and incubated in the absence of S107.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Binding Assay, Concentration Assay, Incubation

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A and B) Skeletal muscle homogenates were incubated without (control) or with 5 mM GSH, 5 mM GSSG or 0.10 mM NOC12 in the absence or presence of 44 µM S107 for 20 h at 24°C. Unbound FKBP12 was removed by centrifugation and the amounts of RyR1 and FKBP12 were detected using anti-RyR1 and anti-FKBP12 antibodies. Homogenates incubated without glutathione and NOC12 served as control. Data are the mean ± SEM of 8 experiments. *p<0.05 compared to control homogenates without S107. # p<0.05 compared to homogenates incubated with NOC12 in the absence of S107.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Incubation, Control, Centrifugation

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A) Immunoblots of RyR1 and FKBP12. SR vesicles depleted of FKBP12 were incubated for 20 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca 2+ , protease inhibitors and 10 nM FKBP12 without or with 44 µM S107 in the absence and presence of 5 mM GSH, 5 mM GSSG or 0.10 mM NOC12. SR vesicles not treated with FK506 and incubated without glutathione served as control. (B) SR vesicles not treated with FK506 served as control. Data are the mean of 5–7 experiments. *p<0.05 compared to FKBP12-depleted vesicles incubated without S107 in the presence of GSH and NOC12, respectively, as determined by paired Student’s t-test.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Western Blot, Incubation, Control

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A) Immunoblots of RyR1 and FKBP12. SR vesicles treated with FK506 were incubated in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca 2+ , protease inhibitors and 1 µM FKBP12 for the indicated times in the presence of 5 mM GSH, and absence or presence of 44 µM S107. FKBP12 binding was stopped by centrifugation. (B) Data are the mean ± SEM of 7 experiments. They were corrected for amounts of FKBP12 associated with FK506 treated SR vesicles kept on ice and normalized to SR vesicles not treated with FK506 (gray bar). *p<0.05 compared to SR vesicles not treated with FK506 and not incubated with FKBP12 and S107. #p<0.05 compared to FKBP12-depleted SR vesicles incubated for the same time (10 min or 2 h) in the presence of FKBP12 but absence of S107.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Western Blot, Incubation, Binding Assay, Centrifugation

Journal: PLoS ONE

Article Title: Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

doi: 10.1371/journal.pone.0054208

Figure Lengend Snippet: (A) SR vesicles not treated (top trace) or treated with FK506 (traces 2 and 5) were incubated for 30 min at 24°C without addition (traces 1 and 2), with 25 µM S107 (trace 3), 5 µM FKBP12 (trace 4) or 25 µM S107 plus 5 µM FKBP12 (bottom trace) in 0.3 M sucrose, 0.25 M KCl, 20 mM imidazole, pH 7.0, and protease inhibitors. Vesicles were then fused to a lipid bilayer and recorded at 2 µM cis cytoplasmic Ca 2+ and −35 mV as described in . Representative single channel currents (downward deflections from closed levels, c–) (left) and current histograms (right) are shown. (B) Single channel data were obtained as described in A. Data are the mean ± SEM of 4–12 single channel recordings. * p<0.05 compared to RyR1 not treated with FK506 in the absence of S107 and FKBP12. # p<0.05 compared to FK506-treated RyR1 incubated in the absence of S107 and FKBP12. % p<0.05 compared to FK506-treated RyR1 incubated with FKBP12 in absence of S107. p values were determined by Student’s t-test.

Article Snippet: FKBP12 monoclonal antibody was from R&D Systems (Minneapolis, MN), anti-Cys-SNO polyclonal from Sigma.

Techniques: Incubation