Chemidoc

The indicated C. difficile strains were grown in TY medium (pH 7.4) supplemented with 2 µg/ml thiamphenicol and 1 µg/ml nisin at 37°C and harvested at H₂₄ (24 h) (74 (link)). Total protein was quantitated using the Pierce Micro BCA protein assay kit (Thermo Scientific), and 8 µg of total protein was separated by electrophoresis on a precast 4 to 15% TGX stain-free gradient gel (Bio-Rad), and total protein was imaged using a ChemiDoc (Bio-Rad). Corresponding gel images for each Western blot are included in the supplemental material as indicated in the text. Protein was then transferred to a 0.45-µm nitrocellulose membrane, and Western blot analysis was conducted with either mouse anti-TcdA (Novus Biologicals) or mouse anti-FLAG (Sigma) primary antibody, followed by goat anti-mouse Alexa Fluor 488 (Life Technologies) secondary antibody. Imaging and densitometry were performed with a ChemiDoc and Image Lab software (Bio-Rad), and a one-way ANOVA, followed by Dunnett’s multiple-comparison test, was performed to assess statistical differences in TcdA protein levels between the rstA mutant and each rstA overexpression strain (GraphPad Prism v6.0). At least three biological replicates were analyzed for each strain, and a representative Western blot image is shown.

2.5 μg oligonucleotides (Sigma) were diluted in 1 × G4 folding buffer (10 mM Tris–HCl pH 7.5, 0.1 M KCl). The samples were incubated at 95 °C for 5 min and let slowly renatured ON at RT; 10 μL were mixed with 2.5 μL 5 × native loading dye and the samples loaded on 15% TBE native gel for about 90 min at 80 V. Note, the gel was pre-run for 15 min at 80 V and the wells rinsed thoroughly. The gel was incubated with 10 μg/mL NMM or ThT in 1 × G4 folding buffer for 15 min under agitation and protected from light. The NMM or ThT signal was detected with a ChemiDoc (Biorad). Then the gel was incubated with 0.5 μg/μL ethidium bromide in 1 × G4 folding buffer for 15 min in agitation to stain the total DNA. The ethidium bromide was detected with a ChemiDoc (Biorad).

The tissue homogenates described in the previous section were used for Western blot analyses. A sample (50 μg) was fractionated by SDS‐PAGE on 7.5 or 12% (w/v) polyacrylamide gels (TGX StainFree FastCast gel; Bio‐Rad Inc., Hercules, CA) and electrophoretically transferred to a polyvinylidene fluoride membrane. The bands in each lane on the membrane were detected with an ultraviolet imager (ChemiDoc; Bio‐Rad). The blots were blocked with 3% (w/v) bovine serum albumin, 1% (w/v) polyvinylpyrrolidone, and 0.3% (v/v) Tween‐20 in PBS for 1 h, and then exposed to a specific primary antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) against Mfn2 (1:500; sc‐100560) or Drp1 (1:500; sc‐271583) diluted in PBS with 0.05% Tween‐20 for 1 h. After the blots had been incubated with an HRP‐labelled mouse IgGκ light chain binding protein (1:5,000; sc‐516102; Santa Cruz), they were reacted with Clarity Western ECL substrate (Bio‐Rad), and the required proteins were detected with an imager (ChemiDoc). The densities of the bands were normalized to the densities of all protein bands in each lane on the membrane (Gilda and Gomes 2013; Vigelsø et al. 2014). The densities of the bands were quantified using Image Lab software (Bio‐Rad) and normalized to the same sample that was run on every gel and transferred to every membrane.

Immunoblots were analyzed as described in Gardezi et al. (2016 (link)). Immunoblots were imaged with the ChemiDoc (Bio-Rad, Hercules, CA, USA) with a broad range of exposure times. For each experiment, protein band intensities were quantified by densitometry from a common blot at a single exposure selected for clear bands without saturation. Background counts were subtracted using an automated rolling disk subtraction. Protein band intensities were normalized to a single control condition for each experiment. For experiments comparing different fusion proteins, intensities were normalized to the C2 fusion protein intensity. For peptide experiments, intensities were normalize to the control peptide condition as described previously (Gardezi et al., 2016 (link)).
Fusion protein concentrations were visualized using Coomassie stain (Sigma-Aldrich) and imaged with the ChemiDoc (Bio-Rad) Coomassie stain protein gel function. Concentrations were quantified by densitometry and background was subtracted using an automated rolling disk subtraction. Fusion protein concentration was used as a loading control so that SV2 (I_SV2) and STG (I_STG) protein intensities were normalized to the fusion protein concentration (I_FP) from the same lane, hence %SV-PD was calculated as I_SV2/I_FP or I_STG/I_FP respectively.

For western blot analysis, cells were washed with cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific, Grand Island, NY, USA). Proteins were quantified using the Bradford method and equal amounts of protein were resolved by SDS-PAGE and analyzed by western blotting as previously described^{33 (link)}. The membranes were incubated with primary antibodies against cleaved PARP, caspase3, survivin (Cell Signaling Technology, Beverly, MA, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C and with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Western blot images were analyzed with a LAS-4000 mini (GE Healthcare Life Sciences, Uppsala, Sweden) and Bio-Rad ChemiDoc (Bio-Rad, Richmond, CA, USA).