Journal: Aging cell

Article Title: Phosphorylation of an RNA-Binding Protein Rck/Me31b by Hippo Is Essential for Adipose Tissue Aging.

doi: 10.1111/acel.70022

Figure Lengend Snippet: FIGURE 5 | Prediction of human RCK structure. (A) Western blot analysis of p-MST1, MST1, HuR, Rck, DCP2, and Actb in cell lysates derived from pre-adipocytes (Day 0) and mature adipocytes (Day 8) from Student's t-test. (B) RT-qPCR levels of Fabp4 and Gcg mRNAs normalized to Gapdh mRNA purified from Rck or IgG immunopellets from cell lysates of precursor and mature adipocytes. Data are expressed as mean ± SD of three in- dependent experiments. N = 3, **p < 0.01, NS, not significant from Student's t-test. (C) Human RCK structure predicted using the AlphaFold database provided by Deepmind and EMBL. The colors indicate the accuracy of the model based on a per-residue confidence score (pLDDT) between 0 and 100. Blue represents pLDDT values over 90 (Very high). Sky blue indicates pLDDT values between 70 and 90 (Confident). Yellow color represents pLDDT values between 50 and 60 (Low). Deep orange color indicates the pLDDT values below 50 (Very low). The phosphorylation sites, Thr 36 and Thr 75, are indicated by the red circles based on the direction of the side chain. (D) mRNA (RT-qPCR, right) and protein (Western blot, left) levels of Fabp4, Gcg, and Actb mRNAs and proteins from pre-adipocytes (Day 0) and mature adipocytes (Day 8) after transfection of the Rck shRNA or control. Values are expressed as mean ± SD of three independent experiments (p < 0.001, Student's t-test). N = 3, ***p < 0.001, NS, not significant from Student's t-test.

Article Snippet: Immunoblots were performed using antibodies targeting Tuba1a (1:5000, SCBT sc- 5286), MST1 (1:1000, CST #3682), p- MST1 (1:500, CST #49332), AUF1 (1:2000, Millipore 07–260), HuR (1:2000, SCBT sc- 5261), QKI (1:1000, Abcam ab126742), HA (1:5000, Sigma 11867423001), hnRNP K (1:1000, SCBT sc28380), EDC3 (1:1000, SCBT sc- 365024), eIF4E (1:1000, SCBT sc- 9976), Rck (1:1000, SCBT sc- 376433), Dcp2 (1:1000, Abcam ab28658), NCL (1:1000, Abcam ab13541), Akh (Drosophila, 1:200, generated by Dr. KS Lee), Act5c (Drosophila, 1:3000, Developmental Studies Hybridoma Bank, University of Iowa, JLA20), Actb (1:5000, SCBT sc- 47778), acetyl- CoA carboxylase 1 (ACC1) (1:1000, CST #4190), fatty acid- binding protein 4 (FABP4) (1:1000, CST #13368), and fatty acid synthase (FASN) (1:1000, SCBT sc- 55580).

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR, Purification, Residue, Phospho-proteomics, Transfection, shRNA, Control

Journal: International Journal of Molecular Sciences

Article Title: Severe Muscle Deconditioning Triggers Early Extracellular Matrix Remodeling and Resident Stem Cell Differentiation into Adipocytes in Healthy Men

doi: 10.3390/ijms23105489

Figure Lengend Snippet: Expression of adipogenic differentiation markers in muscle from VL biopsies before (Pre-DI) and after (Post-DI) 5 days of DI. ( a ) Protein levels of key adipogenic markers in Pre-DI and Post-DI vastus lateralis biopsies. Data were normalized to the total protein staining using Stain-free technology ( b ) Protein levels of two mature adipocyte markers in Pre-DI and Post-DI vastus lateralis biopsies; data were normalized to the total protein staining using Stain-free technology. n = 16/time point for C/EBPβ, FABP4; n = 14/time point for C/EBP⍺, Perilipin; n = 12/time point for PPARg ( c ) Index Fat Content individual percent of Vastus lateralis measured by MRI before (Pre-DI) and after (Post-DI) 5 days of DI. * p < 0.05, Paired t -test or Wilcoxon matched-pairs signed-rank test was performed according to normality.

Article Snippet: , FABP4 , #3544 , 1/500 , Cell Signaling.

Techniques: Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Severe Muscle Deconditioning Triggers Early Extracellular Matrix Remodeling and Resident Stem Cell Differentiation into Adipocytes in Healthy Men

doi: 10.3390/ijms23105489

Figure Lengend Snippet: Western Blot/Immunostaining antibodies.

Article Snippet: , FABP4 , #3544 , 1/500 , Cell Signaling.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Ablation of KDM2A Inhibits Preadipocyte Proliferation and Promotes Adipogenic Differentiation

doi: 10.3390/ijms22189759

Figure Lengend Snippet: KDM2A is localized in the nucleus. ( A ) Immunohistochemical staining of KDM2A in iWAT and eWAT. ( B , C ) Immunofluorescence to detect KDM2A and FABP4 proteins by KDM2A antibody (red) and FABP4 antibody (green), respectively. Nuclei visualized with DAPI (blue) in mice inguinal white adipocytes ( B ) and epididymal white adipocytes ( C ). A merged image is shown in the panel on the far right. DM 13d: the cells were treated by differentiation medium for 13 days. Scale bar: 20 µm. NC represents negative control, IHC represents immunohistochemical staining, HE represents hematoxylin–eosin staining.

Article Snippet: Cells were incubated with a primary anti-KDM2A antibody (Abcam, EPR18602, UK), mouse anti-FABP4 antibody (Bioss, bsm-51247M, Shanghai, China), anti-Ki67 rabbit pAb (Wanlei, WL01384a, Shenyang, China) and anti-PPARγ rabbit pAb (Wanlei, WL01800, China) at 1:500 dilution in a blocked solution at 4 °C overnight.

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Negative Control