Fig 1: Validation of commercially available antibodies for PANK1 and PANK2. (A) Commercially obtained and genomically validated CRISPR knockout HAP1 cell lines were used as negative controls to verify the specificity of the PANK antibodies. Western blot confirming the loss of signal of PANK1 and loss of PANK2 in HAP1 PANK1 and PANK2 CRISPR knockout clones purchased from Horizon Discovery. Rabbit anti-PANK1 mAb from CST (#23887S) correctly identifies a single band at 50 kDa (expected molecular weight of PANK1 protein) in WT HAP1 cells, and the band is absent in the PANK1 KO CRISPR clone. Origene mouse anti-PANK2 (mAb#TA501321) identifies endogenous PANK2 protein in wild type HAP1 cells at the expected size of 48 kD, and the band disappears in the PANK2 CRISPR KO clone. A non-specific band at 37 kD is evident but is sufficiently distinct not to confound interpretation. Triose phosphate isomerase (TPI) was used as a loading control. (B) Validation of CST PANK1 antibody using the PANK1 deleted and PANK1 intact glioma and melanoma lines. G59 (glioma) and 537 MEL (melanoma) are PANK1 homozygous deleted cancer cell lines. (C) Schematic showing the Santa Cruz two-plasmid (gRNA/Cas9/GFP and HDR/Puromycin/RFP) system-mediated CRISPR KO. (D–F). Independent in-house generation of PANK1 and PANK2 CRISPR KO in cancer cells, and validation by Western blot.