Additional information regarding tyrosinated α-tubulin and LYTL tubule elongation. (A) U2OS cells were transfected with a nontargeting control (NTC) or TTL siRNA for 60 h. WB shows tyrosinated α-tubulin, detyrosinated α-tubulin, total α-tubulin, and cyclophilin B protein levels. (B) Histogram showing tyrosinated α-tubulin protein levels normalized to total α-tubulin. Unpaired t test was used (P = 0.066, n = 3). (C) Histogram showing detyrosinated α-tubulin protein levels normalized to total α-tubulin. Unpaired t test with Welch’s correction was used (P = 0.0185, n = 3). (D) Histogram showing the normalized tyrosinated/detyrosinated α-tubulin ratio. Unpaired t test was applied (P = 0.0003, n = 3). Asterisks signal unspecific bands; real bands are shown in arrowheads. (E) U2OS cells were transfected with EMTB-mNeonGreen and treated with DMSO or nocodazole (10 µM, 30 min) and imaged live under a confocal microscope. (E) U2OS cells were treated with DMSO or nocodazole (10 µM, 30 min) and fixed. (F) U2OS cells treated with DMSO or nocodazole were fixed and stained for tyrosinated α-tubulin, detyrosinated α-tubulin, and acetylated α-tubulin. (G and H) U2OS cells were transfected with a NTC or KIF1A, KIF1B, and KIF5B siRNA for 24 h. Cells were then transfected with HaloTag-LRRK2 and mNeonGreen-JIP4 for 36 h and treated with LLOME for 2 h. Cells were then fixed and stained for endogenous LAMP2. (I) Graph depicts the #JIP4⁺ tubules/JIP4⁺ lysosomes ratio in all conditions. Brown–Forsythe and Welch ANOVA with Dunnett’s. Data are mean ± SEM (n = 31 cells). Scale bar = 10 µm. Source data are available for this figure: SourceData FS5.