CRISPR-Cas9 KO of eTNS1 in human erythroblasts does not affect assembly of the membrane skeleton or expression of cytoskeletal proteins. (A) Representative western blots of eTNS1, actin, α1,β1-spectrin, protein 4.1R, GAPDH, α-tubulin, and total protein from nontransfected control and eTNS1-KO erythroid cultures; 15 μg protein loaded per lane (dashed lines indicate blots in which lanes were cropped out). (B) Quantification of immunoblots normalized to total protein. Values are mean ± SD from 4 nontransfected controls and 4 KO cultures. ∗∗P < .01; ∗∗∗P < .001. (C) Single optical sections of Airyscan Z-stacks of human erythroblasts from day-14 eTNS1-KO cultures, depicting stages of enucleation, stained for eTNS1 (red), β1-spectrin (magenta), GPA (green), and nuclei (Hoechst; blue). Scale bar, 5 μm. (D) Single XY optical sections (top panels) of Airyscan Z-stacks of polarized and enucleating eTNS1⁺ and eTNS1⁻ cells were used to construct XZ side views (bottom panels) of each cell at the location of the yellow dashed line in the top panel. (GPA, white; nucleus, blue). eTNS1⁻ cell shapes appeared to resemble biconcave-like discs, with a rim and dimple in some cases), despite the retention of the nucleus in the cell. Scale bar, 5 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, non-significant.