Figure 2. TRIP-Br1 promotes AC1 degradation.

(a) Stable expression of TRIP-Br1-V5 markedly reduced total and cell-surface protein levels of endogenous AC1 in HEK293T cells. Cell-surface AC1 was biotinylated and isolated. Transferrin receptor: loading control. Quantification of the western blots is shown at the top: **, different from the control cell (CTRL), p=0.0053; *p=0.048; n = 3 independent experiments. (b) Knocking down TRIP-Br1 with two different shRNAs (shTRIP-Br1-1 and shTRIP-Br1-2) increased AC1 protein expression in HEK293T cells. Actin: loading control. (c) Knocking out TRIP-Br1 in mice elevated AC1 expression in heart tissue (n = 8, p=0.01), and adipocytes (n = 4, p=0.049), and the brain (n = 3, p=0.011). (d) Changes in the expression of endogenous AC1 with or without shTRIP-Br1-1 treatment were examined for 12 hr after CHX treatment in HEK293T cells (upper). The results are quantified in the lower panel; *, different from control (CTRL), p=0.013 (6 h); **p=0.004 (12 h); n = 3. (e) Effect of proteasomal and lysosomal inhibitors on endogenous AC1 in HEK293T cells. Baf, bafilomycin A1; CQ, chloroquine diphosphate. Quantification of the western blots is shown at the top: *, different from the control (CTRL), p=0.029; n = 3 independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.28021.004

Figure 2—figure supplement 1. Effect of TRIP-Br1 on the expression of other membrane proteins and AC1 mRNA levels.

(a–b) Stable expression of TRIP-Br1-V5 did not affect the protein levels of HA-tagged TRPV4 (transient receptor potential vanilloid 4) (a) and TRPP2 (transient receptor potential polycystin 2) (b) transiently expressed in HEK293T cells. GAPDH, loading control; GFP, transfection efficiency control. (c) AC1 mRNA levels showed no change (compared to control) in the heart tissue of TRIP-Br1 knockout mice in a real-time PCR assay (WT n = 5, KO n = 4; p=0.853). The mRNA level of AC1 was normalized relative to that of GAPDH.

DOI: http://dx.doi.org/10.7554/eLife.28021.005