Figure 2.

Defective E1 Activity of UBA5 in Fibroblasts Derived from Subjects with Pathogenic Biallelic UBA5 Variants

(A) Quantitative real-time PCR analyses of UBA5, UFC1 (MIM: 610554) and UFM1 (MIM: 610553) in case (A-4 and B-3) and control (C1: female, age 26; C2: female, age 43) primary skin fibroblasts. Using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science), cDNA was synthesized from 1 μg of total RNA extracted from indicated fibroblasts. Quantitative PCR was performed using LightCycler 480 Probes Master (Roche Applied Science) in a LightCycler 480 (Roche Applied Science). Signals were assessed relative to that of GAPDH (MIM: 138400). Values were normalized to the amount of mRNA in control C1. The experiments were performed three times. The sequences of the primers are shown in Table S7. Statistical analysis was performed using the unpaired t test (Welch test). Data are means ± SE. ^∗∗p < 0.01.

(B and C) Immunoblot analysis of UBA5, UFC1, and UFM1 with reducing and nonreducing samples that were prepared from fibroblasts of affected individuals and human controls (B) and mouse embryonic fibroblasts (C). Indicated fibroblasts were lysed with ice-cold TNE buffer (10 mM Tris-HCl [pH 7.5], 1% Nonidet P-40, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], and protease inhibitors). Samples were prepared with NuPAGE-loading buffer in presence or absence of DTT, separated using a NuPAGE system (Life Technologies) on 4%–12% Bis-Tris gels in MOPS-SDS buffer, and then transferred to a polyvinylidene difluoride (PVDF) membrane. Mouse monoclonal anti-actin antibody (Chemicon International cat# MAB1501R), rabbit monoclonal anti-UFM1 antibody (Abcam cat# ab109305, RRID: AB_10864675), anti-UBA5 antibody,⁴ and anti-UFC1 antibody⁴ were used for immunodetection. The immunoreactive bands were detected by LAS-4000 (GE Healthcare UK). In the cases of samples prepared without DTT, the intermediates corresponding to UBA5-UFM1 and UFC1-UFM1 were clearly detected. Bar graphs indicate the quantitative densitometric analyses using Multi Gauge Version 3.2 Image software (Fuji Film) of UBA5, UBA5-UFM1, and UFC1-UFM1 intermediates relative to ACTIN.

Statistical analysis was performed using the unpaired t test (Welch test). The data represent the means ± SE of five separate experiments. ^∗p < 0.05 and ^∗∗p < 0.01.