Fig 1: Detection of UCP1 and mitochondrial complex proteins and the levels of NADH and ATP in TG2+/+ and TG2-/- cells together with PGC1a and pAMPK expressions: (A) Representative Western blots of UCP1, II-SDHB, III-UQCRC2, V-ATPSA of beige cells (n = 3); (B) quantitative analyses of Western blots of UCP1, II-SDHB, III-UQCRC2, and V-ATPSA proteins (n = 3); (C) representative Western blots of UCP1, II-SDHB, III-UQCRC2, and V-ATPSA of preadipocytes (n = 3); (D) quantitative analyses of Western blots of UCP1, II-SDHB, III-UQCRC2, and V-ATPSA proteins of preadipocytes; (E) mitochondrial dehydrogenases activity levels in preadipocytes and differentiated beige cells (MTT assays, n = 3); (F) NADH and (G) ATP content of preadipocytes and beige cells; (H) representative Western blots and quantitative analyses of phospho-AMPKa and PGC-1a in preadipocytes and beige cells. ß-ACTIN was used as a loading control. Columns represent the mean values ± SD. Statistical analyses were performed using Student’s t-test. *, **, and *** indicate statistically significant differences at p < 0.05, p < 0.01, or p < 0.001, respectively. n = 3.
Fig 2: Differentiation of beige cells from TG2+/+ and TG2-/- preadipocytes isolated from the epidydimal fat depots: (A) Typical microscopic view of confluent preadipocytes and differentiated beige cells, images were taken using EVOS FL Cell Imaging System, scale bars represent 200 µm; (B) proliferation capacity of TG2+/+ and TG2-/- preadipocytes, cell density values were determined using Sulforhodamine B assays (n = 3); (C) average size of lipid droplets in beige cells; (D) total lipid content in beige cells; (E) gene expression value of the preadipocyte marker (Pref1) and beige marker genes (Ucp1, Tbx1, Tnfrsf9, and Tmem26) in preadipocytes and beige cells (n = 3). Columns represent the mean values ± SD. Statistical analyses were carried out using GraphPad Prism 7.0 version, by two-way ANOVA (Tukey’s multiple comparison test and Student’s t-test. ** p < 0.01, *** p < 0.001.
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