Hrp conjugated anti mouse antibody

The cell lysates were run on a 10% SDS polyacrylamide gel and transferred to a PVDF-Star transfer membrane (AppliChem). The amount of IRF3 and IRF7 in the cell lysates was detected using a mouse anti-V5 antibody (1:5000, Invitrogen), MAVS and cGAS was detected with a mouse anti-FLAG antibody (1:2000, Sigma Aldrich), STING was detected with a rabbit anti-STING antibody (1:1000, Cell signaling), pTBK-1 was detected with a rabbit anti‐pTBK1 (Ser172) (1:1000, Cell Signaling), vinculin was detected with anti-vinculin (Sigma) and the amount of GAPDH was detected using a rabbit anti-GAPDH antibody (1:1000, Santa Cruz Biotechnology). The primary antibodies were followed by either HRP-conjugated anti-mouse antibody (Jackson Immuno research) for the anti-V5 antibody or HRP-conjugated anti-rabbit (Dako Cytomation) for the anti-GAPDH antibody. The western blot was developed using SuperSignal West Dura extended Duration substrate (Thermo Scientific) and visualized on an X-ray film (Konica Minolta).

Lysate buffers (60 mM Tri-HCl, pH 6.8; 50 % glycerol; 2 % SDS; 0.1 % bromophenol blue) contain protease cocktail (cOmplete, mini, EDTA-free protease inhibitor; 1183617001, Millipore Sigma) and phosphatase inhibitor cocktail (P0044, Millipore Sigma). The lysates were precleared of debris by centrifugation at 10,000 g in a refrigerated microcentrifuge for 10 min. Supernatants were mixed with 5 % 2-mercaptoethanol (Millipore Sigma) and then boiled for 10 min. Antibodies used: Rabbit anti--Tubulin III (1:4000, Millipore Sigma); HRP conjugated anti-rabbit antibody (1:10,000, #111-035-045, Jackson ImmunoResearch); Rabbit-anti-SARM1 (1:1000, #13022, Cell signaling) and (1:5000); mouse anti-GFP (1:1000, #2955 S, Cell signaling); HRP-conjugated anti-mouse antibody (1:5000, 115-035-003, Jackson ImmunoResearch).

Brains samples were homogenised in NP-40 lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCL [pH 7.5]) at 10% weight/volume, and subsequently treated at 37 °C for 1 h with proteinase K (20 µg/ml). Samples were then separated by electrophoresis and electroblotted onto polyvinylidene difluoride membranes as described previously^{42 (link)}. PrP was detected using anti-mouse PrP mAb (clone 7A12; Yin et al., 2007), followed by HRP-conjugated anti-mouse antibody (Jackson Immunoresearch, Ely, UK) and visualised with BM Chemiluminescent substrate kit (Roche, Burgess Hill, UK). An uncropped image of the immunoblot is provided in Supplementary Fig. S1.

A similar approach was used as described above. MaxiSorp immuno plates (Nunc) were coated with 20 nM WT D1_401-560 overnight at 4°C. The next day, the wells were washed and blocked with PBSTB buffer (PBS containing 0.1% Tween-20 and 2% BSA) before being incubated with 16 nM truncated wild-type TcdB or mutated TcdB (containing a His-tag at the C-terminus). After incubation, wells were washed 4 times with PBST. Bound TcdB was detected using a mouse anti-His antibody (0.5 μg/mL, R&D Systems, catalog #MAB050-SP) and an HRP-conjugated anti-mouse antibody (0.8 μg/mL, Jackson ImmunoResearch, catalog #: 115-035-146).

Third instar larvae or S2 cells were lysed with lysis buffer and boiled with sample loading buffer at 95 °C for 10 mins [39 (link)]. Prepared samples were loaded on 7.5% SDS-PAGE gel, and protein bands in the gel were transferred to nitrocellulose membrane by wet method. Membranes were blocked with 5% dry milk in TBS-T (140 mM NaCl; 3 mM KCl; 25 mM Tris pH 7.4; 0.1% Tween-20) at room temperature (RT) for 1 h. Membranes were sequentially probed in primary antibody and HRP-conjugated secondary antibody solution diluted with 2% dry milk in TBS-T. After washing, membranes were developed with WESTSAVE-Gold reagent (AbFrontier).
Primary antibodies used are: concentrated anti-Wg (mouse, 1:2000, Developmental Studies Hybridoma Bank (DSHB)), anti-GFP (rabbit, 1:10,000, abcam), anti-αTub (mouse, 1:5000, Sigma-Aldrich), anti-MYC (rabbit, 1:5000, abcam) and anti-Ago (guinea pig, 1:5000, gift from K. H. Moberg). Secondary antibodies used are: HRP-conjugated anti-mouse antibody (1:10,000, Jackson Laboratory), HRP-conjugated anti-rabbit antibody (1:10,000, Jackson Laboratory) and HRP-conjugated anti-guinea pig antibody (1:5000, Jackson Laboratory).

To assess the $\mathit{\beta }$ -catenin accumulation in HeLa cells, cells were
treated with Wnt3a in the presence of Dvl inhibitors for 24 h and lysed in
RIPA buffer (50 mM Tris, pH 8.0, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS, 150 mM NaCl). Equal amounts of protein were loaded on a SDS-PAGE. Separated proteins were blotted onto PVDF membranes (polyvinylidene fluoride) for immunoblot analysis using anti- $\mathit{\beta }$ -catenin antibody (610154, BD). HRP-conjugated anti-mouse antibody (715-035-150, Jackson ImmunoResearch Laboratories) was used for secondary detection with Western lightning chemiluminescence reagent plus (PerkinElmer) and Vilber Lourmat imaging system SL-3.