Generation of INPP5B ^{Degron/Degron} cells. (A) Schematic representation of the chicken inpp5b genomic locus and the strategy used to knock in an auxin degron tag at the C-terminus. Exons are shown in black boxes. Two sequential rounds of transfections with targeting constructs bearing different antibiotic resistance genes were performed. bsd, Blasticidin; pac, Puromycin. The antibiotic selection cassettes were removed from the genome by Cre recombination. (B) RT-PCR analysis of WT DT40 cells and INPP5B^{Degron/Degron} cells using gene-specific primers flanking the inpp5b stop codon. (C) Cell-free extracts from WT DT40 cells and INPP5B^{Degron/Degron} cells that were treated with or without auxin for 2 h were subjected to IP using anti-FLAG antibody. INPP5B was detected by WB using anti-poly His. (D) Representative confocal images at the indicated time points from INPP5B^{Degron/Degron} cells stimulated with anti-IgM antibody in the presence of LysoTracker Green to visualize lysosomes. Colocalization events are denoted by blue arrowheads. Source data are available for this figure: SourceData FS1.