Figure 3. Cholesterol analogue (CHS) stabilises LAT1-CD98 during affinity purification.

(A) LAT1 was purified by immunoaffinity precipitation utilising the V5 epitope from HEK293S GnTIˉ cells stably expressing pcDNA3.1 LAT1-V5-6xHis. Representative SEC profiles detected by absorbance at 280 nm are shown. (B) The peaks eluted at 8.4 mL (1), 9.5 ml (2) and 10.5 ml (3) from the SEC profile with CHS were analysed by immunoblotting for CD98 in non-reducing conditions. (C) Peak 3 from SEC was concentrated, run on SDS-PAGE then coomassie stained or immunoblotted for the His₆ tag and CD98 in non-reducing conditions. (D) Stability of the purified LAT1-CD98 at 4 °C was monitored for up to 7 days after purification by analytical SEC. (E) The purified LAT1-CD98 was heated for 10 minutes at the indicated temperatures and run on analytical SEC. (F) The effect of the heat stress on the purified LAT1-CD98 was quantified from the chromatograms by calculating the normalised absorbance (n = 3). The normalised absorbance of the purified LAT1-CD98 was plotted against temperature with the melting point determined (T_m ± standard error).