Western Blot Analysis of KLC Proteins

Methods were modified from those previously described^{3 (link),24 (link)}. Samples were mixed with SDS buffer (125 mM Tris pH 6.8; 2% SDS; 5% 2-beta mercaptoethanol; 5% glycerol; with bromophenol blue). After boiling, samples were subjected to SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose membranes (Protan BA85, Whatman). After blocking non-specific sites with 5% skimmed milk protein, membranes were incubated with primary antibody: Rabbit pAb KLC1 [N2C2] (GeneTex, Cat No: GTX114510) or rabbit pAb KLC1 (Abcam, Cat No: ab187179), or the following mouse mAbs: KLC (H7) (Santa Cruz, Cat No: sc-515792), KLC (63–90) (Prof. Scott Brady^{43 (link)}, University of Illinois at Chicago), or MANEM1 5D10 against emerin^{44 (link)}, MANLAC1 4A7 against Lamin A/C^{45 (link)}, or mAb GST 17A10 (raised against recombinant GST). This was followed by washing and incubation with either peroxidase-labelled goat anti-rabbit immunoglobulins (Dako, Denmark, Cat No: P0448) or peroxidase-labelled rabbit anti-mouse immunoglobulins (Dako, Cat No: P0260) (1/1000). Antibody reacting bands were detected with SuperSignal West Femto chemiluminescent detection system (ThermoFisher Scientific, Cat No: 34094) and ChemiDoc Touch imaging system (BioRad Ltd.).