FIGURE 4.

CLPX is essential for heme-dependent degradation of ALAS1. A, along with the induction of ALAS1F, siNegative control (siNC), siCLPX-1 (siCLPX), or siCLPP-1 (siCLPP) RNA or no siRNA (cont.) was introduced into FT293^ALAS1F cells. Then ALAS1F protein was immunoprecipitated as described under “Experimental Procedures.” After immunoprecipitation, equal volumes of immunoprecipitates (FLAG-IP) or equal amounts of total cell lysate (10 μg/lane; cell lysate) were loaded for the detection of each protein using SDS-PAGE followed by immunoblotting analysis. FLAG-tagged ALAS1 protein, CLPX, or CLPP was detected in immunoprecipitates and cell lysate. GAPDH was also detected in the total cell lysate as a loading control. B, target sequence of Cas9 in the human CLPX gene and the DNA sequence of the flanking region of the CLPX gene in FT293^ΔCLPX. The red underline and open red box of the reference sequence (WT) indicate the target sequence of Cas9 and protospacer adjacent motif (PAM), respectively. C, expression of CLPX and ALAS1 after incubation with or without doxycycline. GAPDH was detected as a loading control. D, effect of CLPX expression on the decrease in ALAS1 after hemin treatment. The influence of hemin treatment was determined in FT293 cells (lanes 1 and 2) or CLPX^ind/FT293^ΔCLPX without (lanes 3 and 4) or with (lanes 5 and 6) induction of CLPX expression by doxycycline. p or m, precursor or mature FLAG-tagged ALAS1 protein, respectively.