Luminol kit

Transfected HeLa p62 KO cells were harvested in 50 mM Tris, pH 7.4, 2% SDS, and 1% glycerol. Cell lysates were cleared by centrifugation, and supernatants resolved by SDS-PAGE and transferred to Hybond-ECL nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% nonfat dry milk in PBS-T, incubated with primary antibody overnight, and HRP-conjugated secondary antibody for 1 h at room temperature. Proteins were detected by immunoblotting with a chemiluminescence Luminol kit (SC-2048, Santa Cruz Biotechnology) using a LumiAnalyst Imager (Roche Applied Sciences).

Image Lab software (Bio-Rad) was used to quantify T3SS cargo protein bands relative to those of DMSO-treated controls. The WT Y. pseudotuberculosis YopE, P. aeruginosa ExoU, or S. enterica SipA, SipC, FliC, and FliD bands in DMSO control samples were set to 1.00. To evaluate type III secretion of ExsE in P. aeruginosa, Western blotting against T3SS cargo was carried out using a polyvinylidene difluoride (PVDF) membrane (Millipore). Prior to blocking, membranes were incubated with acetone at 4°C for 30 min with gentle shaking. Membranes were then moved to Tris-buffered saline with 0.1% Tween 20 (TBST) and heated to 50°C for 30 min. Blots were blocked in 2.5% nonfat milk for 1 h at room temperature and incubated with anti-ExsE at 4°C overnight with gentle shaking. Blots were washed three times for 5 min each in TBST. Horseradish peroxidase conjugated secondary antibody was then incubated for 1 h at room temperature. Signals were detected with a luminol kit (catalog number sc-2048; Santa Cruz Biotechnology, Inc.) after washing. ExsE, BSA, RpoA, and SipC were visualized with anti-ExsE antibody (courtesy of Timothy Yahr) (20% tris-tricine gel), anti-BSA (catalog number 2A3E6; Santa Cruz Biotechnology, Inc.), anti-RpoA (gift from Melanie Marketon) (7.5% tris-glycine gel), and anti-SipC (catalog number ABIN335178; Antibodies-online, Inc.) (10% tris-glycine gel), respectively.

The protocol included tissue homogenisation with a pestle and mortar in ice-cold RIPA buffer (Amresco) and protease inhibitor cocktail (Sigma-Aldrich), sonication for 1 h at 4 °C, spinning for 30 min at 14,000 rpm at 4 °C and denaturing at 95 °C for 5 min. NuPAGE Novex 4–12% Bis-Tris protein gels (Invitrogen, UK) were used for separation. After transfer to PVDF membranes (Invitrogen, UK), samples were incubated in 5% non-fat milk powder (Sigma, UK) for 1 h at room temperature, then in anti-Na_v1.7 and anti-β-tubulin III antibodies at 4 °C overnight followed by incubation in secondary antibodies at room temperature for 1 h and visualisation with the Luminol kit (Santa Cruz, USA; Supplementary Table 2). Membranes were examined in a G:Box (SynGene, UK) using the GeneSnap software package (Synoptics Ltd, SynGene). Analysis was done by ImageJ; Na_v1.7 intensities were normalised to β-tubulin intensities in each of the 16 samples (samples from the ipsilateral and contralateral sides of 8 animals). Then, the ratio of the normalised intensities found on the ipsilateral and contralateral sides in each animal was calculated and averaged.