Journal: Cell reports

Article Title: The GSK-3β-FBXL21 Axis Contributes to Circadian TCAP Degradation and Skeletal Muscle Function

doi: 10.1016/j.celrep.2020.108140

Figure Lengend Snippet: (A) Y2H test showing that FBXL3 and FBXL21 interact with TCAP. Positive control yeasts were transformed with the pGBKT7-p53 bait plasmid and the pGADT7-SV40 Tag prey plasmid. pGBKT7-53 encodes the Gal4 DNA binding domain (BD) fused with murine p53; pGADT7-T encodes the Gal4 activation domain (AD) fused with SV40 large T antigen. (B) Interaction of FBXL3 and FBXL21 with TCAP. 293T cells were transfected with FLAG-Fbxl3, FLAG-Fbxl21, and hemagglutinin (HA)-TCAP, and immunoprecipitation was performed using anti-FLAG antibody (M2). (C and D) FBXL21, but not FBXL3, decreases the TCAP amount in a dose-dependent manner. 293T (C) and C2C12 (D) cells were co-transfected with the indicated constructs. The graphs below show quantification of the dose effects of FBXL3 and FBXL21 on TCAP stability using unnormalized optical density (OD) values, and error bars represent ± SEM (n = 4). TCAP abundance shows a significant statistical difference between TCAP alone and different FBXL21 concentrations in 293T and C2C12 cells (t test, **p < 0.01, ***p < 0.001, ****p < 0.0001). (E) Top left panel: FBXL21 showed anti-phase oscillation with TCAP in skeletal muscle. Right panels: quantification of FBXL21 and TCAP levels using unnormalized OD values. Error bars represent ± SEM (n = 3). Two-way ANOVA shows a significant statistical difference between TCAP and FBXL21 (p < 0.0001). Bonferroni’s multiple comparisons test shows a significant difference at ZT16 (p < 0.05) and ZT20 (p < 0.01). One-way ANOVA with Tukey’s post hoc analysis shows a statistically significant differences in the FBXL21 amount between time points (*p < 0.05). Bottom left panel: co-immunoprecipitation showing circadian time-dependent TCAP-FBXL21 interaction. (F) Differential localization of FBXL3 and FBXL21 in 293T cells. Scale bars, 20 μm. See also .

Article Snippet: For endogenous TCAP expression detection from skeletal muscle and C2C12 samples, we used anti-TCAP antibody (BD).

Techniques: Positive Control, Transformation Assay, Plasmid Preparation, Binding Assay, Activation Assay, Transfection, Immunoprecipitation, Construct

Journal: Cell reports

Article Title: The GSK-3β-FBXL21 Axis Contributes to Circadian TCAP Degradation and Skeletal Muscle Function

doi: 10.1016/j.celrep.2020.108140

Figure Lengend Snippet: (A) The five lysine sites in TCAP were individually mutated to arginine, and the stability of each K-to-R mutant was examined by co-transfecting the Fbxl21 expression construct into 293T cells. (B) Impaired FBXL21-mediated degradation of the TCAP K26R/K98R mutant. Immunoblotting was performed to detect TCAP (~25 kD) and FBXLs using anti-FLAG antibody. (C) Quantification of the effect of FBXL21 on WT TCAP and K26R/K98R stability. Error bars represent ± SEM (n = 3). Half-life: TCAP, 5.5 h, TCAP/FBXL21, 2.3 h, TCAPK26R/K98R, 19.4 h, TCAPK26R/K98R /FBXL21, 9.4 h). (D) TCAP oscillation in skeletal muscle from WT and Psttm mutant mice. Immunoblotting was performed using total protein extracts with TCAP antibody. Representative blots from three independent experiments are shown, and quantification is shown in the right panel (error bars represent ± SEM, n = 3). One-way ANOVA with Tukey’s post hoc analysis shows statistically significant differences in the TCAP amount between time points in WT (*p < 0.05) but not Psttm mice. Two-way ANOVA shows significant statistical differences between WT and Psttm mice for TCAP expression (p < 0.01). Blue and red circles represent WT and Psttm mice, respectively. (E) Real-time RT-PCR analysis of TCAP mRNA expression in WT (blue) and Psttm (red) mice. Two-way ANOVA shows a significant statistical difference for TCAP mRNA at ZT0 and ZT12 (error bars represent ± SEM, n = 3; ****p < 0.0001) but no significance between WT and Psttm mice. See also .

Article Snippet: For endogenous TCAP expression detection from skeletal muscle and C2C12 samples, we used anti-TCAP antibody (BD).

Techniques: Mutagenesis, Expressing, Construct, Western Blot, Quantitative RT-PCR

Journal: Cell reports

Article Title: The GSK-3β-FBXL21 Axis Contributes to Circadian TCAP Degradation and Skeletal Muscle Function

doi: 10.1016/j.celrep.2020.108140

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For endogenous TCAP expression detection from skeletal muscle and C2C12 samples, we used anti-TCAP antibody (BD).

Techniques: Recombinant, Kinase Assay, Mutagenesis, Software

Journal: Human Reproduction (Oxford, England)

Article Title: Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation

doi: 10.1093/humrep/deab032

Figure Lengend Snippet: Confirmation of podocalyxin (PCX) expression in human endometrial epithelial cells (HEECs). (A) Schematic illustration of the protein domain organization of human PCX. SP, signal peptide; TM, transmembrane domain. Numeric numbers on the top indicate amino acids (aa) at domain boundaries. Thin bars at the bottom indicate antigen regions used to raise the three antibodies, antibody 1 (Ab1, 23-427 aa), antibody 2 [Ab2, 282-558 aa, the darker part (aa 282-444) indicates the mapped epitope], and antibody 3 (Ab3, 300-500 aa). (B) Western blot analysis of PCX protein using the three antibodies shown in (A). Lane 1, recombinant human PCX (the extracellular domain only). Lane 2, total lysates of human umbilical vein endothelial cells (HUVECs) as positive control. Lane 3, total lysates of primary HEECs isolated from the proliferative phase endometrial tissue. β-actin used as a loading control. A representative of 4 independent experiments is shown. (C) Real-time RT-PCR analysis of PCX mRNA expression in HUVECs (n = 4) and HEECs (n = 5). Data normalized to 18S rRNA.

Article Snippet: The sections were incubated for 1 h at 37 °C with a primary PCX antibody (Ab2, 2 μg/ml, SC-23904 3D3; Santa Cruz, USA) in 10% v/v FBS in high salt TBS containing 0.1% v/v Tween20.

Techniques: Expressing, Western Blot, Recombinant, Positive Control, Isolation, Control, Quantitative RT-PCR