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. Author manuscript; available in PMC: 2017 Aug 10.
Published in final edited form as: Cell Rep. 2017 Jan 3;18(1):93–106. doi: 10.1016/j.celrep.2016.12.015

Figure 4. VSTM2A Is a Glycoprotein Secreted by Committed Preadipocytes.

Figure 4

(A) Western blot analyses of cell lysates and culture media of 293T cells overexpressing V5-VSTM2A. Cells were plated the day before and culture medium was changed at time 0. Cells were lysed and culture medium was collected at the indicated time. S6K was used as a loading control. The images are representative of two independent experiments.

(B) Western blot analyses of cell lysates and culture media of 293T cells overexpressing V5-VSTM2A and treated or not with Brefeldin A (3 μg/mL). S6K was used as a loading control. The images are representative of three independent experiments.

(C) Western blot analyses of cell lysates and culture media of 293T cells overexpressing V5-VSTM2A and treated or not with tunicamycin (1 μg/mL). Cells were plated the day before and pre-treated with tunicamycin for 6 hr. S6K was used as a loading control. The images are representative of three independent experiments.

(D) Western blots analyses showing that VSTM2A is a glycoprotein. Lysates and media isolated from 293T cells overexpressing V5-VSTM2A were treated with PNGase F following manufacturer’s instructions (NEB, P0407) and analyzed by western blotting. S6K was used as a loading control. The images are representative of two independent experiments.

(E) Western blot analyses of cell lysates and culture media of 293T cells overexpressing wild-type (WT) V5-VSTM2A or V5-VSTM2A with asparagine to glutamine (N→Q) mutations of N35, N175, or N35+N175. S6K was used as a loading control. This experiment was performed once.

(F) Western blots analyses showing that VSTM2A dimerizes following its secretion. Media was isolated from 293T cells overexpressing V5-VSTM2A was kept on ice or denaturated by heat and/or β-mercaptoethanol. This experiment was performed three times.

(G) Western blot analyses of cell culture media of NIH 3T3, a low adipogenic 3T3-L1 line (4H4) a high adipogenic 3T3-L1 line (4D2) and parental 3T3-L1 cells. Cells were plated until confluence and then the culture medium was changed (time 0), collected over 3 days, and analyzed for endogenous VSTM2A. This experiment was performed three times with the same outcome.

(H) Western blot analysis of cell lysate and culture media collected from eWAT explants. Samples of eWAT were collected from P4 mice and incubated in DMEM 10% for the indicated time.

See also Figure S4.