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. 2024 Jul 19;300(8):107562. doi: 10.1016/j.jbc.2024.107562

Figure 5.

Figure 5

p.S141C variant is retained in the ER due to the additional Cys residue and forms a disulphide-linked complex. A, fluorescence micrographs of HeLa cells expressing WT and p.S141C SBP-HaloTag-leptin before biotin addition, and after biotin at the indicated time points. Representative of n= 7 for WT SBP-HaloTag-leptin, n = 3 for p.S141C SBP-HaloTag-leptin. Scale bar: 10 μm. B, HeLa cells expressing WT or S141C SBP-HaloTag-leptin were treated with biotin for indicated times and the HaloTag fluorescence in cell lysate (top) and culture media (bottom) measured. n = 2. C, kinetic secretory analysis of 3T3-L1 adipocytes expressing WT or p.S141C SBP-HaloTag-leptin treated with biotin where indicated. Cells were imaged at 20 min time intervals from 0 to 240 min. HaloTag fluorescence intensity was normalised to the 0 min time point. n = 3 for WT SBP-HaloTag-leptin; n = 2 for p.S141C SBP-HaloTag-leptin. D, kinetic secretory analysis of HeLa cells expressing WT, p.S141C or p.S141A SBP-HaloTag-leptin treated with biotin where indicated. Cells were imaged at 20 min time intervals from 0 to 240 min. HaloTag fluorescence intensity was measured using an image analysis pipeline and normalised to 0 min time point. n = 2 for WT and p.S141C SBP-HaloTag-leptin; n = 3 for p.S141A SBP-HaloTag-leptin. E, leptin variant molecular weight analysis by Western blot under reducing and non-reducing conditions. HeLa cells were treated with NEM to alkylate free cysteines and prevent disulfide bond formation or exchange after cell lysis. Anti-HaloTag antibody was used to detect SBP-HaloTag-leptin. A higher molecular weight anti-Halo-Tag antibody reactive band in cells expressing the p.S141C variant is indicated. GAPDH abundance was assessed as loading control. Data are representative of n = 4.