Figure 6.

Glutamic acid 73 of PLIN2 negatively affects PLIN2 binding to adiposomes

Adiposomes were prepared according to Transparent Methods. PLIN2 and its mutants were expressed in Transetta (DE3). The bacteria were cultured in the same condition. Each 800 ml of bacteria suspension was collected and removed the medium. The bacteria were mixed with 10 ml Tris-NaCl buffer (50 mM Tris-HCl, 150 mM NaCl, pH = 7.4) to resuspended and sonicated on ice for 15 min (6 s on and 6 s off) using a probe with a power of 210 W, to prepare the bacterial homogenate. The bacterial lysate was obtained by collecting 1 ml homogenate to perform centrifugation at 21,130g for 10 min, and the supernatant was the lysate. Thirty μl of adiposomes (OD600 = 20) was incubated with 10 μl of bacterial lysate containing PLIN2 or mutants, the adiposomes were reisolated and bound proteins were determined using Western blot with anti-PLIN2 antibody. The density of protein band was quantified by ImageJ from three independent experiments.

(A) Western blots of adiposome-bound PLIN2 and its mutants in the (1) absence or (2) presence of PtdIns using anti-PLIN2 antibody.

(B) Comparative quantification using ratio of protein band in lane 2 versus lane 1 within the same mutant. Data represent mean ± s.e.m., n = 3. ∗p< 0.05, two-tailed t-test.

See also Figures S4 and S5, Tables S1 and S2.