recombinant fkbp12  (R&D Systems)

Journal: PLOS ONE

Article Title: Ca 2+ -PP2B-PSD-95 axis: A novel regulatory mechanism of the phosphorylation state of Serine 295 of PSD-95

doi: 10.1371/journal.pone.0313441

Figure Lengend Snippet: (A) Western-blot analysis of the in-vitro dephosphorylation assay using mouse brain extracts. The “Control” sample (lane 1) was not subject to the dephosphorylation assay. (B) Quantification of dephosphorylation in vitro (pS295/PSD-95) incubated with calcium (Ca 2+ (+)) or without calcium (Ca 2+ (-)), relative to no-incubation (Control) conditions. The data are represented as the mean ± standard deviation (n = 3 from three independent experiments). *** P < 0.001 by the unpaired Student’s t -test; n.s., not significant. (C, D) Western-blot analysis (C) and quantification (D) of the efficiency of suppression by different doses of FK506 on the Ca 2+ -dependent dephosphorylation of pS295 in vitro . (E, F) Western-blot analysis (E) and quantification (F) showing that coapplication of FK506 and FKBP12 efficiently suppressed the Ca 2+ -dependent dephosphorylation of pS295 in vitro . The data are represented as the mean ± standard deviation overlaid with individual data points (n = 3 from three independent experiments). *** P < 0.001 by two-way ANOVA with the post-hoc Tukey’s multiple comparison test; n.s., not significant. F (1,16) = 383.3, P < 0.0001 for FKBP12+FK506, F (3,16) = 48.44, P < 0.0001 for Ca 2+ , and F (3,16) = 47.06, P < 0.0001 for interaction.

Article Snippet: NMDA was obtained from Sigma-Aldrich (St. Louis, MO, USA), EGTA from Wako Chemicals (Osaka, Japan), FK506 from ChemScene (Monmouth Junction, NJ, USA), ascomycin from Cayman Chemical (Ann Arbor, MI, USA), cyclosporine A from Nacalai Tesque (Kyoto, Japan), calyculin A from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and recombinant FKBP12 from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, In Vitro, De-Phosphorylation Assay, Control, Incubation, Standard Deviation, Comparison

Journal: Journal of molecular and cellular cardiology

Article Title: Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level

doi: 10.1016/j.yjmcc.2024.01.003

Figure Lengend Snippet: Quantification of endogenous and overexpressed FKBP12.6 in mouse hearts. A, Immunoblots of FKBP12.6 from left ventricular (LV) homogenates of WT mice. A GST-tagged recombinant protein (RP-FKBP12.6-GST) was used to quantify precisely FKBP12.6 expression. Fifty μg of total proteins were loaded for each WT sample. B, Linear regression of the standard curve of the RP-FKBP12.6-GST absorbance measured on the immunoblot showed in panel A. C, Immunoblots of FKBP12.6 in WT and TG mice with 10 μg of loaded proteins from each LV homogenates and four concentrations of RP-FKBP12.6-GST. D, Quantification of FKBP12.6 expression in LV homogenates from WT and both TG mouse lines ( N = 4 mice for each). Statistical analysis was conducted with One-way ANOVA (P ANOVA = 3.772e-6) followed by post-hoc Sidak test (P S ). E, Immunoblots of FKBP12.6 in LV homogenates after loading 1.5 μg and 75 μg of proteins from TG and WT LV homogenates, respectively.

Article Snippet: Then, we used our custom-made specific anti-FKBP12.6 antibody (Eurogentec, rabbit, 1/1000) [ ], whose specificity was verified by using a commercial FKBP12 recombinant protein with a C-terminal His-tag (SinoBiological, #10268-H08E) of 12.9 kDa ( Fig. S1A and ).

Techniques: Western Blot, Recombinant, Expressing

Journal: Journal of molecular and cellular cardiology

Article Title: Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level

doi: 10.1016/j.yjmcc.2024.01.003

Figure Lengend Snippet: FKBP12.6 overexpression did not modify RyR2 phosphorylation before and following β-adrenergic stimulation. A, RyR2 protein expression level in TG1 and TG2 mouse lines, with immunoblot (top) and protein quantification (bottom), expressed as median [5–95%] in whisker boxes. Protein levels were normalized to β-actin and expressed as fold-change of that determined in WT mice ( N = 5–6). Non-parametric Kruskal-Wallis test was used (P KW = 0.0222) followed by Dunn’s test. B, Immunoblot (top) and quantifications (bottom) of RyR2 phosphorylation at the PKA site (Ser2808), and, C, at the CaMKII site (Ser2814) normalized to the total RyR2 in cardiomyocytes, in WT, TG1, and TG2 ( N = 5–9), in basal conditions and under Iso stimulation (100 nM). ANOVA of aligned rank transformed data was used ((B) Ser2808: P genotype = 0.0002; P treatment = 0.0101; P interaction = 0.1928; (C) Ser2814: P genotype = 0.4541; P treatment = 0.6341; P interaction = 0.3539).

Article Snippet: Then, we used our custom-made specific anti-FKBP12.6 antibody (Eurogentec, rabbit, 1/1000) [ ], whose specificity was verified by using a commercial FKBP12 recombinant protein with a C-terminal His-tag (SinoBiological, #10268-H08E) of 12.9 kDa ( Fig. S1A and ).

Techniques: Over Expression, Expressing, Western Blot, Whisker Assay, Transformation Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level

doi: 10.1016/j.yjmcc.2024.01.003

Figure Lengend Snippet: FKBP12.6 overexpression mitigated proarrhythmic SR Ca 2+ waves and stress-induced bidirectional ventricular tachycardia in RyR2 R420Q CPVT mice. A, ECG of CPVT (top) and DT (bottom) mice before (left) and under Epi/Caff stimulation (i.p. 2 and 120 mg/kg, respectively; right). B, Percentage of mice with bidirectional ventricular tachycardia (BDVT) episodes under Epi/Caff stimulation, shown with colour in CPVT (pink; N = 4) and DT (purple; N = 5) mice. C, Duration of BDVT during Epi/Caff stimulation in CPVT (pink squares; N = 4) and DT (purple diamonds; N = 5) mice. Non-parametric Mann-Whitney test was used. D, Line-scan confocal images of caffeine-evoked [Ca 2+ ] i transients in CPVT RyR2 R420Q (top) and DT (bottom) myocytes, with above each image the fluorescence trace. The thick line above each image indicates 10 mM caffeine perfusion, after the 2 Hz cell stimulation. E, Peak of the [Ca 2+ ] i transients expressed as F/F 0 , and F, time constant of the [Ca 2+ ] i transient decay in CPVT (pink squares; N = 4 mice; n = 51 cells) and DT myocytes (purple diamonds; N = 4 mice; n = 48 cells). ANOVA of aligned rank transformed data was used. G, Peak of the caffeine-evoked [Ca 2+ ] i transients (expressed as F/F 0 ) in CPVT (pink squares; N = 4 mice; n = 22 cells) and DT (purple diamonds; N = 4 mice; n = 18 cells) myocytes. ANOVA of aligned rank transformed data was used. H, Line-scan confocal images of Ca 2+ waves in quiescent CPVT-RyR2 R420Q (top), and DT (bottom) myocytes. I, Ca 2+ wave frequencies in basal conditions in CPVT (pink squares; N = 4 mice; n = 41 cells) and DT (purple diamonds; N = 4 mice; n = 43 cells) myocytes. ANOVA of aligned rank transformed data was used. J, Arrhythmic diastolic events (Ca 2+ waves + triggered activities) in CPVT in basal conditions (pink squares; N = 4 mice, n = 19 cells) and after incubation with 1 μM JTV-519 (red wine squares; N = 3 mice; n = 20 cells). ANOVA of aligned rank transformed data was used. All data are shown as median [5–95%] in whisker boxes.

Article Snippet: Then, we used our custom-made specific anti-FKBP12.6 antibody (Eurogentec, rabbit, 1/1000) [ ], whose specificity was verified by using a commercial FKBP12 recombinant protein with a C-terminal His-tag (SinoBiological, #10268-H08E) of 12.9 kDa ( Fig. S1A and ).

Techniques: Over Expression, MANN-WHITNEY, Fluorescence, Cell Stimulation, Transformation Assay, Incubation, Whisker Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level

doi: 10.1016/j.yjmcc.2024.01.003

Figure Lengend Snippet:

Article Snippet: Then, we used our custom-made specific anti-FKBP12.6 antibody (Eurogentec, rabbit, 1/1000) [ ], whose specificity was verified by using a commercial FKBP12 recombinant protein with a C-terminal His-tag (SinoBiological, #10268-H08E) of 12.9 kDa ( Fig. S1A and ).

Techniques:

Journal: Nature

Article Title: Brain-restricted mTOR inhibition with binary pharmacology.

doi: 10.1038/s41586-022-05213-y

Figure Lengend Snippet: Fig. 1 | RapaLink-1 is a potent mTOR inhibitor that requires FKBP12 for its cellular activity. a, Chemical structures of RapaLink-1 and FK506. b, Inhibition of mTOR activity by MLN128, RapaLink-1 or rapamycin in the presence or absence of 10 µM FKBP12 in the in vitro kinase assay (n = 2; data are plotted as individual points). c, K562 CRISPRi cells transduced with sgRNAs targeting GAL4-4 (control) or FKBP12 were treated with RapaLink-1 and cell proliferation was assessed after 72 h. In the last listed condition, cells were transduced with

Article Snippet: Expression and purification of recombinant FKBP12 DNA sequences encoding full-length human FKBP12 were synthesized by Twist Biosciences and cloned into the pET47b vector using standard molecular biology techniques.

Techniques: Activity Assay, Inhibition, In Vitro, Kinase Assay, Transduction, Control