GGTA1 Protein Analysis in Piglet Pancreas

Protein extraction and immunoblotting were performed as previously described in our previous study [25 (link)]. The pancreas tissue from GTKO piglets and cloned piglet derived from unmodified original donor cells were used to evaluate GGTA1 protein levels using western blotting. In brief, pancreas tissues were lysed in RIPA lysis buffer (Bestbio, China) with protease inhibitors at 4 °C. After lysis, the supernatants were obtained by centrifugation at 13,800 × g for 15 min at 4 °C. Equal amounts of protein (70 μg) were run on SDS-PAGE gel, along with molecular weight marker. After electrophoresis, the proteins were transferred to PVDF membranes and reacted with primary antibodies against GGTA1 (ALX-801-090-1, Enzo, Lausen, Switzerland; 1:15) and β-actin (anti-β-actin, Sigma-Aldrich; 1:2000) at 4 °C overnight. After incubation, the membranes were washed and incubated with anti-mouse secondary antibodies (R&D, USA). The membranes were incubated with the ECL (Easysee Western Blot Kit, China) and visualized with an Imaging System (Bio-Rad, Universal Hood II, USA).