Fig. 4: CD99L2 is ubiquitinated, and degraded via autophagy and proteasomes.
From: Loss-of-function variants in the CAPN1 activator CD99L2 cause X-linked spastic ataxia
a Western blot of myc-His6-CD99L2 in 293T and SH-SY5Y cells using CD99L2- and myc tag-specific antibodies. β-actin served as loading control. Grey arrowheads indicate modified full-length (flmo) CD99L2, white arrowheads unmodified full-length (fl) CD99L2, and black arrowheads a breakdown product (bdp). b, c Western blots of K48-linked polyubiquitin (K48-polyUb), LC3B-I/II, and CD99L2 in 293T cells expressing myc-His6-CD99L2 after proteasomal (MG132) or autophagosomal (bafilomycin A1, BafA1) inhibition. β-actin served as loading control. d Quantification of modified and full-length CD99L2 after treatment. n = 4 biologically independent experiments. One-sample t- or Student’s t-tests. e, f Ubiquitination analysis of myc-His6-CD99L2 by ubiquitin-directed immunoprecipitation (IP: Ub) in MG132-treated cells. Total- and K48-linked ubiquitin was detected (e). CD99L2 was detected with an N-terminal antibody (f). GAPDH served as loading control. Grey arrowheads indicate ubiquitinated (Ub) CD99L2. LE, long exposure; SE, short exposure. g Schematic of lysine residue distribution within CD99L2. SP signal peptide, ECD extracellular domain, TMD transmembrane domain, CPD cytoplasmic domain. h Western blot of wild-type (WT) and lysine-free (K0) CD99L2 variants (14 lysine residues mutated to arginine) in 293T cells detected with a myc tag-specific antibody. GAPDH served as loading control. i Quantification of WT and K0 CD99L2. n = 6 biologically independent experiments. One-sample t-test. Bars represent mean ± s.e.m. All t-tests are two-tailed. P values are shown in the graphs. Source data are provided as a Source Data file.
