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. 2021 Nov 8;22(21):12079. doi: 10.3390/ijms222112079

Figure 1.

Figure 1

AMBRA1 regulates the self-association, but not the expression, of ALDH1B1. (A) The interaction between Flag-tagged AMBRA1 (Flag-AMBRA1) and Myc-tagged ALDH1B1 (Myc-ALDH1B1) was identified by a co-immunoprecipitation (co-IP) assay in HEK293T cells. α-Tubulin was detected as a loading control (n = 5). (B) Protein expression of Myc-ALDH1B1 after overexpression of indicated amounts of 3×F vector (pCMV-3Tag-6, empty vector) and Flag-AMBRA1, as measured by immunoblot analysis. The 3×F vector and Flag-AMBRA1 were transfected into HCT116 cells that stably overexpressed Myc-ALDH1B1 (pCMV-Myc-ALDH1B1 HCT116 cells) (n = 3). (C) The interaction between Flag-tagged ALDH1B1 (Flag-ALDH1B1) and Myc-tagged ALDH1B1 (Myc-ALDH1B1) was confirmed by immunoprecipitation and immunoblot analysis (n = 3). (D) The empty vector and Flag-AMBRA1 were cotransfected with Flag-ALDH1B1 into pCMV-Myc-ALDH1B1 HCT116 cells. Then, co-IP was performed using anti-c-Myc agarose affinity gel antibody and western blotting was conducted using anti-Flag, anti-Myc, and anti-α-tubulin antibodies (n = 3). (E) Quantification of the results in (D) using the ImageJ software. Protein levels of Flag-ALDH1B1 in immunoprecipitate samples were normalized to that of Myc-ALDH1B1. The ratio of the control (3×F) was set at 1. ** p < 0.01, *** p < 0.001 vs. control.