Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis
Fig 2
Verification of expression, secretion, activity and localization of TsUBE2L3.
A. T.sp SP, HEK 293T cell lysate and T. spiralis muscle larvae lysate were separated by SDS-PAGE and immuno-blotted with anti-HsUBE2L3 and anti-tubulin antibodies. B. Auto-ubiquitination of parkin with Ub-biotin was probed by streptavidin-HRP blot. Reactions from left to right: lane 1: human E1 (UBE1A), E3 (parkin) and Ub-biotin only (no E2). Reactions in lanes 2–4 included human E1, parkin and Ub-biotin, with the following E2 substitution: lane 2: T.sp SP after resin-bound BSA depletion (BSA), lane 3: T.sp SP after resin-bound anti-tubulin depletion (α-tubulin), lane 4: T.sp SP after resin-bound α-HsUBE2L3 depletion (α-HsUBE2L3). In lanes 5–7 the elution fractions from the depletions shown in lanes 2–4 were used to substitute E2. C. The pixel intensity of lanes 2–7 of the depletion assay (B) was analysed using an ImageJ gel analysis plugin. For each of the three samples tested in the assay (BSA, α-tubulin and α-HsUBE2L3) the sum of the pixel intensity of the depletion plus elution lanes was taken as 100% (intensity of lane 2 + 5 for BSA and so on). The relative percentages of the depletion and elution lanes were then calculated (intensity of lane 2/(2+5) x 100) and plotted. D. A TsUBE2L3-specific antibody was made and its specificity was assessed by immuno-blot against T.sp L1 lysate, T.sp SP and rat (R.n) skeletal muscle tissue (SMT) lysate. The arrow indicates the expected size of TsUBE2L3. E. T. spiralis infected rat skeletal muscle tissue (R.n SMT) was sectioned and analyzed by immuno-histofluorescence. A single T.sp L1 inside the nurse cell (n), surrounded by a collagen capsule (c) is displayed. Tissue was probed with anti-TsUBE2L3 antibodies (Alexa-568, red) and nuclei were stained using DAPI (blue). Brightfield (BF)/DAPI/anti-TsUBE2L3 and DAPI/anti-TsUBE2L3 merged images are displayed. As a control, infected R.nSMT was probed with PBS and DAPI only. Arrows indicate stacks of stichocyte cells in the stichosome. F. Annotated diagram of T. spiralis L1 morphology. Image adapted and reprinted from Trichinosis in Man and Animals by S.E. Gould under a CC BY license, with permission from Charles C. Thomas Publisher LTD, original copyright 1970.