Figure 1. Confirmation of successful transfection of the 2 missense succinic semialdehyde dehydrogenase (SSADH) alleles in HEK293 by Western blot and reverse transcription PCR (RT-PCR).

Cell pellets were resuspended in urea lysis buffer (10 mmol/L Tris HCl, 8 mol/L urea, 100 mmol/L NaCl, pH 8.0) and the protein content was determined using the bicinchoninic acid method (Sigma-Aldrich, St Louis, MO). Thirty micrograms of total protein was size-separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 12% stain‐free TGX gel (Bio‐Rad Laboratories, Hercules, CA). Proteins were transferred to a polyvinylidene fluoride membrane using the Trans‐Blot Turbo Transfer System (Bio‐Rad). Immunodetection was performed using the ALDH5A1 mouse polyclonal antibody (B01P; Abnova Corporation, Taipei, Taiwan), anti-mouse secondary antibody (A9044; Sigma-Aldrich, St Louis, MO), and enhanced chemiluminescent substrates (Lumi-Light Plus Western Blotting Substrate; Roche Applied Science, Indianapolis, IN). Images were acquired and analyzed with the ChemiDoc MP imager and Image Lab software (Bio-Rad). Total RNA was isolated from the HEK293 transfectants and cDNA was synthesized using standard molecular biology techniques. mRNA expression of the transfected constructs is confirmed by RT-PCR amplification of the full length cloned ALDH5A1 open reading frame, with the forward primer specific for the pAD1/RSV ALDH5A1 cDNA sequence.¹⁹ *Lower molecular weight protein band detected for the p. Gly441Arg construct, which could be due to the instability and partial degradation of the mutated protein, but alterations in posttranslational protein modification cannot be excluded.