Multi gauge version 3.2 image software

Cells were lysed with ice-cold TNE buffer (10 mM Tris-Cl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and protease inhibitors). The samples were separated using the NuPAGE® system (Invitrogen) on 12% Bis-Tris gels in NuPAGE® MOPS SDS Running Buffer, and transferred to polyvinylidene difluoride (PVDF) membranes. Antibodies against FLAG (Medical & Biological Laboratories Co., Ltd., M185-3L), UFC (Abcam, ab189251) and UFM1 (Abcam, ab109305) were purchased from the indicated suppliers. Anti–UBA5 and UFM1 polyclonal antibodies were described previously (Komatsu et al., 2004 (link)). The immunoreactive bands were detected by LAS-4000 (GE Healthcare UK Ltd.). The quantitative densitometric analyses of FLAG-UBA5-MYC-UFM1, FLAG-UFC1-MYC-UFM1, endogenous UBA5-UFM1 intermediate, and endogenous UFC1-UFM1 intermediate relative to free FLAG-UBA5, FLAG-UFC1, endogenous UBA5, and endogenous UFC1 were carried out using Multi Gauge Version 3.2 Image software (Fuji Film, Tokyo, Japan). Statistical analysis was performed using an unpaired t-test (Welch test). The data represent the means ± standard error (SE) of three separate experiments.

Cells were lysed with ice-cold TNE buffer (10 mM Tris-Cl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], and protease inhibitors). The samples were separated using the NuPAGE system (Invitrogen) on 12% Bis-Tris gels in NuPAGE MOPS SDS Running Buffer and transferred to polyvinylidene difluoride (PVDF) membranes. Monoclonal antibody against FLAG was purchased from Medical & Biological Laboratories Co., Ltd., M185-3L. The immunoreactive bands were detected by LAS-4000 (GE Healthcare UK Ltd.). The quantitative densitometric analyses of FLAG–UBA5–MYC–UFM1ΔC2 relative to free FLAG–UBA5 and FLAG–UFC1–MYC–UFM1ΔC2 relative to free FLAG–UFC1 were carried out using Multi Gauge Version 3.2 Image software (Fuji Film). Statistical analysis was performed using an unpaired t-test (Welch test). The data represents the means ± SE of three separate experiments.