All Xenopus procedures and studies were conducted in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance #A-3216-01). The following procedures have been previously described (Edens and Levy, 2014a (link), 2016 (link)). X. laevis egg extracts and sperm nuclei were used to assemble nuclei in vitro. Reactions were supplemented with ∼28 nM recombinant wt or mutant GFP-LB3 protein 45 min after initiating nuclear assembly. Egg extract nuclei were isolated and resuspended in late-embryo extract to induce nuclear shrinking. For immunofluorescence, nuclei in egg or embryo extract were fixed, spun onto coverslips, and processed for immunofluorescence. A primary rabbit antibody that recognizes phospho–PKC α/β (bs-3333R; Bioss USA) was used at a dilution of 1:100. For LB3 staining, we used a previously described rabbit LB3 antibody at 1:500 (Levy and Heald, 2010 (link)). For NPC staining, we used mAb414 (Covance) at 1:1000. Secondary antibodies were Alexa Fluor 488 or 568 anti-mouse or anti-rabbit immunoglobulin G (Molecular Probes) used at 1:1000.