Fig. 5. MFSD7C and heme regulate thermogenesis through SERCA2b in THP-1 cells.
a 293FT cells were transfected with HA-tagged murine MFSD7C and FLAG-tagged murine SERCA2b. Thirty hours later, MG132 was added into half of the cells and the other half was not treated. Another 35 h later, some cells were treated with 40 μM heme for 1 h before lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody, and eluted, followed by anti-HA immunoprecipitation and elution. Total cell lysate and elute were subjected to Western blotting and probed with anti-SERCA2b or anti-MFSD7C antibodies. Shown are representative data from one of three experiments. b 293T cells were co-transfected with HA-MFSD7C and FLAG-SERCA2b. Twenty-four hours later, cells were either not treated or treated with 10 μM MG132 for either 6 or 12 h. The cells were lysed and subjected to FLAG pull-down and blotted with anti-MFSD7C, anti-SERCA2b and anti-ubiquitin antibodies. Shown are representative data from one of three experiments. c SERCA2b protein levels in parental THP-1 cells, 7CKO cells (3D12, A11, B11, and 4B8) or SERCA2b−/− (#1 and #3) cells. Parental THP-1 cells were either not treated or treated with heme before lysis. Shown are representative data from one of four experiments. d THP-1, 4B8, 4B8FL, and 4B8ΔN cells were either not treated or treated with 40 μM heme for 1 h, lysed and subjected to Western blotting with anti-MFSD7C (top), anti-SERCA2b (middle), and anti-®-tubulin (bottom). Shown are representative data from one of two experiments. e THP-1 cells were incubated with FPT for 6 h, washed, treated with or without 4 μM thapsigargin for 2 h. Cells were washed and treated with 40 μM heme for 1 h before flow cytometry. Representative FPT histograms from one of three experiments are shown. f Parental and Serca2b−/− THP-1 cells were incubated with FPT for 6 h, washed and treated with or without 40 μM heme for 1 h, followed by flow cytometry. Shown are representative FPT histograms from one of three experiments.