Ab59013

In this study, we used the following antibodies for Western blotting and co-immunoprecipitation experiments: rabbit anti-ALAD (1:1000, Abcam ab59013); rabbit anti-ALAS1 (1:500, Abcam ab84962); rabbit anti-FECH (1:500, Abcam ab137042); mouse anti-TOMM20, (1:2000, Abcam ab56783); mouse anti-β-Tubulin, (1:2000, Sigma T8328); mouse anti-α-Tubulin, (1:2000, Sigma T5168); rabbit anti-HSC20, (1:50 for Co-IP experiments, Sigma HPA018447); mouse anti-HSC20, (1:1000, OriGene TA507285); mouse anti-FLAG Tag, (1:100 for Co-IP experiments, Genscript A00187); rabbit anti-MT2A, (1:200, LifeSpan Bio LS-C667906-50); mouse anti-FLAG, (1:2000, OriGene TA50011-100); rabbit anti-ISCU antibody, (1;1000, prepared in our lab).

The protein level was detected by following the previously described protocol (Lv et al., 2020 (link)). In detail, whole embryos of alas2 or alad mutants and their siblings at 36 hpf were collected for Alas2 and Alad protein level detection using anti-Alas2 (1:2000, GTX127887, GeneTex) or anti-Alad (1:2000, ab59013, Abcam) antibody, respectively. The flow cytometric-sorted 30,000 HSPCs (kdrl⁺/cmyb⁺) of control, alas2^−/− or alad^−/− at 36 hpf were used for detection of Fth1, Gpx4, Slc7a11 and Tfr1b protein levels using anti-Fth1 (1:2000, A19544, ABclonal), anti-Gpx4 (1:1000, A1933, ABclonal), anti-Slc7a11 (1:1000, A15604, ABclonal) and anti-Tfr1b (1:2000, 10084-2-AP, Proteintech) antibodies, respectively.