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. 2022 Apr 12;219(5):e20212491. doi: 10.1084/jem.20212491

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© 2022 Yin et al.

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Figure 6. — FNIP1 affects SERCA activity and modulates intracellular Ca²⁺ homeostasis. (A) Left: Western blot analysis confirms the expression of Flag-tagged FNIP1 and HA-tagged SERCA2b in indicated cells. Right: SERCA ATPase activity in HEK293T cell homogenates. n = 3 independent experiments. (B) Ca²⁺-transporting activity of microsomes prepared from HEK293T cells expressing the indicated proteins. n = 4 independent experiments. (C–E) Ca²⁺ transients in HEK293T cells expressing mCherry-SERCA2b together with or without FNIP1 protein. Ca²⁺ dynamics were monitored using Fluo-4 AM in cells following exposure to 100 nM ATP. n = 81–85 cells per group from four independent experiments. Quantitative analysis of the Fluo-4 AM fluorescence intensity (F_t/F₀; C). Quantitation of amplitude (D), FDHM, and time constant Tau (E) of Ca²⁺ transients. (F) HEK293T cells were co-transfected with plasmids encoding HA-SERCA2b, Flag-FNIP1, or control vector. Left: CHX treatment of cells for the indicated time. Right: Quantification of the SERCA2b protein levels. n = 3 independent experiments. (G) Top: Western blot analysis of SERCA2 protein expression in iWAT of indicated male mice. Bottom: Quantification of SERCA2/Tubulin signal ratios. n = 10–12 mice per group. (H) SERCA ATPase activity in iWAT homogenates from male WT and FNIP1 AKO mice. n = 4–5 mice per group. (I) SERCA ATPase activity in cell homogenates from differentiated white adipocyte isolated from iWAT of WT and FNIP1 KO mice. n = 3 independent experiments. (J) Ca²⁺-transporting activity of microsomes prepared from differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. n = 4 independent experiments. (K–M) Ca²⁺ transients in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Ca²⁺ dynamics were monitored using Fluo-4 AM in cells following exposure to 100 nM ATP. n = 62–72 cells per group from three independent experiments. Quantitative analysis of the Fluo-4 AM fluorescence intensity (F_t/F₀; K). Quantitation of amplitude (L), FDHM, and time constant Tau (M) of Ca²⁺ transients. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P value was determined using two-tailed unpaired Student’s t-test. Data are representative of three (A, F–I, and K–M) or four independent experiments (B–E and J). Source data are available for this figure: SourceData F6.