Srx 101a medical film processor

20μg of protein was separated on an SDS-polyacrylamide gel (5% GLI2, 7.5% GLI3) for 30min at 80V followed by 90min at 100V. Gels were transferred to an Immuno-Blot PVDF membrane (Bio-Rad; Cat #1620177), blocked with western blocking buffer (30g BSA, 2ml 10% NaN3, Q.S. 1L TBST) for 5min, and probed with antibody diluted in western blocking buffer. Membranes were washed for 3x5min in TBST and then probed with secondary antibody for 1h at RT. Membranes were then washed 12x5min at RT. Protein was detected by fluorescence, using ECL Primer Western Blotting Detection Reagents (RPN2232) developed on a Konica Minolta SRX-101A Medical Film Processor. All primary and secondary antibodies are listed in S3 and S4 Tables, respectively.

Protein assay was performed using the Pierce Rapid Gold BCA Protein Assay kit (Thermo Fisher Scientific) on snap frozen lung lysate or BAL fluid. 25 ug of lysate or equal volumes of BAL fluid (to add 25 ug of the most concentrated sample) were prepared for each of the antibodies to be used in Bolt LDS sample buffer and Bolt sample reducing agent (Thermo Fisher Scientific) and denatured at 70°C for 10 minutes. Samples and SeeBlue Plus2 pre-stained protein ladder were run on Bolt Bis-Tris 4–12% gel (Thermo Fisher Scientific) and transferred to nitrocellulose. Blots were rinsed with water, imaged and de-stained with 1X tris buffered saline with 0.05% Tween 20 (TBS-T). Blots were then blocked with 5% milk and incubated with primary antibody followed by secondary antibody. Immunoblots were exposed to Super Signal West Pico plus or Fempto chemiluminescent substrate (Thermo Fisher Scientific) and exposed to Amersham HyperFilm ECL (Fisher) film for 1 second to 5 minutes and developed using SRX-101A Medical Film Processor (Konica Minolta). The following primary antibodies were used: caspase-4 (Clone: 4B9; Santa Cruz), caspase-8 (90A992; Novus Biologicals); and GSDMDC1 (64-Y; Santa Cruz). Separate gels were run for each of the antibodies using the same sample preparation on the same day with the same loading control.

The integration of the deletion and reconstitution cassettes into the sglA locus was investigated by Southern blot analysis. WT, ΔsglA, and ΔsglA::sglA⁺ conidia were grown in 10 mL of YUU medium at 37°C overnight with agitation. DNA was isolated as described previously by Malavazi and Goldman (37 (link)), with a few modifications. Mycelium disruption was achieved by lyophilizing frozen suspensions instead of grinding them with a mortar and pestle. The 5′-UTR sequence used as a probe was amplified from the genomic DNA of ΔKu80 pyrG⁻ with the Southern blot 5′ fw and rv primers (Table S3). HindIII-digested DNA fragments were run on a 1% agarose gel overnight at 15 V and transferred to a nylon membrane as described previously by Singh et al. (52 (link)). Radioactive labeling of the probe with [α-³²P]dCTP (Perkin-Elmer) was performed using a Random Primers DNA labeling system kit (Invitrogen) according to the manufacturer’s instructions. The membrane was exposed to Carestream BioMax MR film (Millipore-Sigma), and bands were developed using the SRX-101A medical film processor (Konica Minolta).

Samples were separated by SDS‐PAGE and transferred onto a PVDF membrane (Millipore™) by electroblotting at 100 mA for 75 min. The membranes were blocked with 3% (w/v) milk in 1xTBS overnight at 4°C with shaking followed by 1h incubation with primary antibody (TasA (1:25,000 v/v) as indicated) diluted in 3% (w/v) milk in 1x TBS. Production of the TasA antibody has been previously described (Ostrowski et al., 2011). This was followed by 3 washes of 10 min each with 1x TBS and 2% (v/v) Tween20 and subsequent 45 min incubation with goat anti‐rabbit conjugated secondary antibody (1:5000 v/v) (Pierce™). Membrane was washed 3 times for 10 mins with TBST then developed by ECL incubation and exposing to X‐ray film (Konica™) using the Medical Film Processor SRX‐101A (Konica™). This is with the exception of the data shown in Fig. 4C which was developed as detailed above and visualised using GeneGnomeXRQ (Synegene™).

HEK293T cells were harvested 48 hours after transfection in Phosphate-Buffer Saline (PBS; Severn Biotech LTD) complemented with phosphatase inhibitors (PhoSTOP; Roche) and proteinase inhibitors (COMPLETE; Roche). LCLs were harvested by collecting cells in 15 mL tube (Falcon), centrifuged to form a pellet and resuspended in PBS complemented with phosphatase inhibitors (PhoSTOP; Roche) and proteinase inhibitors (COMPLETE; Roche). Cells were then lysed and processed as previously described (Scotter et al., 2014 (link)). Membrane imaging was conducted using goat anti-rabbit and anti-mouse IgG (H+L) DyLight 680 Conjugate (cat. no. 35568 and 35521, Thermo Life Sciences) and an LI-COR Odyssey or using horseradish peroxidase secondary antibodies for mice (Millipore, 12-349) or rabbits (Millipore, 12-348) and developed through an Enhanced Chemiluminescence System using Medical Film Processor SRX-101A (Konica Minolta). Western blot quantification was performed using the image analysis software, ImageJ (http://imagej.nih.gov/ij/(Schindelin et al., 2012 (link))).