Figure 5.
A1R activation accelerates SCAP protein degradation through SQSTM1 in lysosome
(A) Relative protein expression of SCAP in the AML-12 cells treated with CPA or DPCPX in the presence of cycloheximide (CHX, 50 μM) for 0, 2, 4, and 8 h.
(B) Relative protein expression of SCAP in the AML-12 cells treated or co-treated with CPA and bafilomycin A1 (Baf.A1, lysosomal inhibitor, 1 μM, 8 h) or MG132 (proteasome inhibitor, 10 μM, 8 h).
(C) SCAP colocalizes with the lysosome. AML-12 cells were treated with DPCPX or CPA for 48 h. Fluorescent staining of nuclei (Hoechst, blue), lysosome (red), and SCAP (green) was performed.
(D) Co-immunoprecipitation (coIP) of PKAc or SQSTM1 with SCAP in the AML-12 cells treated with DPCPX or CPA for 12 h.
(E and F) Representative results and quantification of the proximity ligation assay (PLA) analysis of (E) PKAc or (F) SQSTM1 with SCAP in the AML-12 cells treated with CPA or DPCPX. Results are representative of one biological replicate. Cell experiments performed n = 3. Data are depicted as mean ± SEM. Student’s unpaired t test; ∗p < 0.05, ∗∗p < 0.01.