Hrp conjugated anti mouse antibody

Approximately twenty micrograms of tissue were cut into fragments and placed in 2.0 mL screw cap tube containing 10 mm glass beads (Biospec) in 500 μL of RIPA buffer (Sigma) supplemented with protease inhibitors (Roche, Burgess Hill, UK) were disrupted using an automated tissue homogenizer (Biddy Scientific) for two thirty second pulses. Supernatant was transferred to a clean tube and spun at 14 000 rpm in a refrigerated table top microcentrifuge. Twenty microliters of the supernatant was run on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (GE Healthcare) using a semi-dry transfer unit (Bio-Rad). The blot was blocked in 3% non-fat milk at ambient temperature for one hour on a rotating shaker before incubation with an antibody directed to the IBDV capsid protein VP2 (generated by the microbiological services of The Pirbright Institute) in a 1:500 dilution overnight at 5 °C with gentle agitation. The blot was rinsed and a secondary HRP conjugated anti-mouse antibody (Sigma-Aldrich) was used at 1:10 000 dilution for one hour at room temperature before rinsing and visualization using ECL solution (Millipore) and exposure to X-ray film (GE Healthcare).

Protein extracts from the BMDMs and BMDCs were run on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) Immobilon membranes (Thermo). Rabbit anti-STING, anti-cGAS, anti-phospho-IRF3 (Ser 396), anti-IRF3, anti-TBK1, anti-phospho-TBK1 (Ser 172), and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies, all from Cell Signaling Technology; mouse monoclonal anti-DDX41 (Santa Cruz Biotechnology); and HRP-conjugated anti-mouse antibody (Sigma-Aldrich) were used for detection, using either ECL Western blot detection reagent or ECL prime Western blot detection reagent (GE Healthcare Life Sciences).

Formalin-fixed paraffin-embedded (FFPE) tissue blocks of control and KC epithelium were made and processed for immunohistochemical analysis. Immunohistochemistry (IHC) was performed on Bench Mark GX (Ventana Roche) Automated devices for the Bio-SB antibody at M/s S.M Surgipath, Chennai, using β-Catenin (Abcam-ab22656) and YAP (CST-14074) antibodies. β catenin protein was visualized with HRP conjugated anti mouse antibody (Sigma Aldrich). The peroxidase was detected with 3,3′ diaminobenzidine (Sigma Aldrich) each section was counter stained with Mayer’s Haematoxylin (sigma Aldrich). All stained sections were analysed under a light microscope (Eclipse Ni-U Upright Microscope from Nikon Instruments Inc).

Proteins were extracted from the inflorescences as described above. After protein extracts were quantified using the Bradford assay kit and 30 μg of total protein from each sample was separated on a 12.5 % SDS-PAGE gel (we use the acrylamide/bis solution (37.5:1, 2.6 % C), Carl Roth), proteins were transferred onto a PVDF membrane in the Towbin buffer with a wet blotting system (Bio-Rad), the membrane was then blocked with 5 %(w/v) non-fat dry milk in TBST (20 mM Tris-Cl, pH7.6, 137 mM NaCl, 0.1 %(v/v) Tween 20). To detect CDKA;1 proteins, the membrane was probed with a 1:5000 dilution of anti-PSTAIR monoclonal antibody (Sigma) and 1:10,000 HRP-conjugated anti-mouse antibody (KPL) in TBST. Enhanced chemiluminescence detection was performed with HRP substrate (Millipore). The signals were obtained by exposing a X-ray film.

Cell lysates were prepared by harvesting 2 ml of B. cereus wild type and ΔsecDF mutant cultures by centrifugation at 4500xg for 5 min. The pellets were washed once in cold PBS and stored over night at −20°C. Cell pellets were then resuspended in TES containing 2 mg/ml lysozyme and the volume was adjusted according to the original culture OD. The bacterial suspensions were incubated at 37°C for 1 h. After partial cell wall degradation, cell lysis was achieved by six rounds of freezing and thawing in liquid nitrogen and a 37°C water bath respectively. Cell debris was removed by centrifugation and the supernatant was stored on ice for no more than 4 h. Twenty µl of normalized, sterile-filtered supernatants and 2 µl of cell lysates were separated on 10% SDS polyacrylamide gels and blotted onto a nitrocellulose membrane. Toxin components were detected using 1∶20 dilutions of the following monoclonal antibodies: 1A8 and 1E11, against NheA and NheB, respectively [33] (link); and 1E9 and 8B12, specific for the L1 and L2-subunits of Hbl [32] (link). 1∶10,000 dilution of HRP-conjugated anti-mouse antibody (Sigma) was used for chemiluminescent signal development.