Figure 4: Characterization of loss-of-function and gain-of-function ABHD proteins in ABHD5-null BAs.

Doxycycline-inducible ABHD-mCherry proteins were expressed in a BA cell line in which endogenous ABHD5 was silenced by shRNA. In the immunoblots shown in 4a and 4b, it should be noted that the mCherry tag shifts the ABHD protein molecular weight from ~43 kDa for endogenous ABHD5 to ~72 kDa for the mCherry-tagged protein. (a) ABHD5 R299N and G328S mutations inhibit basal (top panel) and isoproterenol-stimulated lipolysis (middle panel). Values are mean ± SEM from three or more independent experiments. Representative ABHD5 immunoblot is shown below, demonstrating absence of endogenous ABHD5 (~43 kDa) in all cell lines. ***p < 0.05 or < 0.01, respectively, compared to ABHD5 mutant cell lines. (b) Lipolysis activation in BA cell lines expressing ABHD4 or ABHD4 N303R/S332G. Representative dsRed (ABHD protein mCherry tag) immunoblot is shown below. Values are means ± SEM from four independent experiments. BA, lysate from differentiated wild-type BA cells. *p < 0.05 compared to ABHD4. (c) Isoproterenol dose-response of BA cell lines expressing ABHD5 (A5) or ABHD4 N303R/S332G (A4 RG). In order to obtain similar levels of ABHD4 protein expression (Figure S2b), ABHD5 and ABHD4 N303R/S332G BA cells were induced with, respectively, 0.0125 and 1.0 μg/mL doxycycline for 42–48 hrs. Mean ± SEM from four independent experiments is shown. (d) ABHD5 and ABHD4 N303R/S332G BA cell line lipolysis requires ATGL and hormone sensitive lipase. Cells were treated with 10 μM atglistatin or 5 μM BAY 59–9435 in the presence or absence of 100 nM isoproterenol. In 4d, expression of ABHD protein in both cell lines was induced with 1 μg/mL doxycycline, which induces higher expression of ABHD5 than ABHD4 N303R/S332G (Figure S2a). Mean ± SEM from three independent experiments is shown. *p < 0.05; **p < 0.01; ***p < 0.001.