Fig 2: Wild type (WT) splice variant DNA and protein analysis. A: representative schematic of the four WT splice variants of rat Atg16l1. Gray boxes designate exons. B: representative agarose gel images depicting splice variants detected in different tissues. C: summary of WT splice variants detected in various tissues. D: representative schematic of predicted protein isoform for each splice variant. Dotted lines denote the area of each protein isoform missing relative to the full-length protein (Atg16l1-A). shaded box, ATG5 binding motif; diagonal hashmark box, coiled coil domain; vertical hashline box, WD40 repeat domains (7 total). E: representative Western blot image showing protein expression when DNA constructs coding for each splice variant were transfected into HEK293 cells. A, Atg16l1-A; B, Atg16l1-B; C, Atg16l1-C; D, Atg16l1-D; E, nontransfected HEK293 cells (negative control). Ladder: Precision Plus Protein Kaleidoscope Prestained Protein Standards (1610375; BioRad); Loading control: ß-actin.

Fig 3: (A) PCR analysis of two different EcN strains. Chromosomal DNA specific (I) and plasmid specific (II) PCR analyses; Lanes: lane M: Marker (GeneRuler 100 bp DNA Ladder, Thermo Fisher Scientific, Germany, Cat. No. SM0321), lane 1: EcN/pMUT-gfp Knr, lane 2: EcN (wild-type), (B) Analysis of plasmid stability of EcN/pMUT-gfp Knr bacteria after 20 generations with and without kanamycin (Kn) selection. The Kn positive cells of EcN/pMUT-gfp Knr bacteria were presented as mean values of two independent experiments. ± standard deviations in percentages. (C) Growth curve of EcN pMUT-gfp strain. At different time points, the optical density of the bacterial culture was measured at 600 nm (OD600) and the bacterial number was presented as a logarithm value (log). The measuring time points are represented as dots (D) Western blot analysis of cultures of EcN or EcN/pMUT-gfp Knr bacteria at different time points. Lane M: protein marker (Precision Plus Protein™ Kaleidoscop, Bio-Rad, Germany, Cat.No.1610375), lane 1: EcN (t = 120 min; OD600 = 0.16), lane 2: EcN (t = overnight; OD600 = 2.54), lane 3: EcN/pMUT-gfp Knr, (t = 0 min; OD600 = 0.001), lane 4: EcN/pMUT-gfp Knr (t = 60 min; OD600 = 0.017), lane 5: EcN/pMUT-gfp Knr (t = 120 min; OD600 = 0.145), lane 6: EcN/pMUT-gfp Knr (t = 180 min; OD600 = 0.813), lane 7: EcN/pMUT-gfp Knr (t = 220 min; OD600 = 1.271), lane 8: EcN/pMUT-gfp Knr (t = 260 min; OD600 = 1.564), lane 9: EcN/pMUT-gfp Knr (t = 300 min; OD600 = 1.803), lane 10: EcN/pMUT-gfp Knr (t = 330 min; OD600 = 1.943), lane 11: EcN/pMUT-gfp Knr (t = overnight; OD600 = 2.646), lane M. Notes: t = 0, start of bacterial growth after inoculation. The GFP protein has a molecular weight of ~27 kDa. The results show a double band around 25 to 27 kDa.

Fig 4: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a) and western blotting (b) of rP32Trx protein. Line 1: Total cell lysate of Escherichia coli BL21(DE3) containing pET32a/P32, 6 h incubation after isopropyl-ß-D-1-galactopyranoside induction; Line 2: purified P32 protein (500 mM imidazole); Lane M: Molecular weight markers (#1610375, BioRad).