Photographic film

Proteins from lysed cells were separated on polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Milipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in wash buffer (20 mM Tris–HCl, pH 7.4, 140 mM NaCl, 0.1% Tween 20) and incubated with anti-alpha smooth muscle actin (α-SMA) [59 (link)] (obtained from C. Chaponnier, Geneva, Switzerland) or anti-vimentin (Dako, Baar, Switzerland) antibodies diluted in blocking solution. Following three washes, membranes were incubated with peroxidase-conjugated goat anti-mouse antibodies (Molecular Probes Inc, Eugene, OR, USA) diluted 1:6000 in wash buffer. Proteins were revealed by chemiluminescence (ECL, Interchim Inc., Montluçon, France) on photographic film (GE healthcare, Chicago, IL, USA) and signals were quantified using the Quantity One software (PDI, Inc., Huntington Station, NY, USA) and normalized by the expression of GAPDH or vimentin.

After incubation cells or tissue were lysed in sample buffer containing 50 mmol/L Tris-HCl pH 6.8, 2% w/v sodium dodecyl sulfate, 10% glycerol, 0.0025% w/v bromophenol blue, and 5% β-mercaptoethanol. Protein samples were then loaded and resolved on Mini-PROTEAN TGX Gels and then transferred onto polyvinylidene fluoride membranes (BioRad) using a Trans-Blot Turbo Transfer System (BioRad). The polyvinylidene fluoride membranes were then blocked with 10% nonfat dry milk or BSA in PBS with 0.1% v/v Tween (PBS-T) for 1 hour, and then incubated with the primary antibodies. Membranes were developed with the Pierce ECL detection system (Thermo-Fisher Scientific) and signal detected using photographic film (GE Healthcare, catalog no. 28-9068-37) and an automatic processor (Fuji RG II). Samples were immunoblotted for Phospho-VASP (Ser239; Cell Signaling, No. 3114), Phospho-VASP (Ser157; Cell Signaling, no. 84519), cGAS (Invitrogen, no. PA5-76367), Phospho-TBK1 (Ser172; Cell Signaling, no. 5483), TBK1 (Cell Signaling, no. 3013), Phospho-STING (Ser365; Cell Signaling, no. 72971), CD31 (Abcam, no. ab28364), MRP1 (Santa Cruz, no. sc-18835), LRRC8A (Santa Cruz, no. sc-517113), GAPDH (Cell Signaling, no. 2118), and β-actin (Sigma-Aldrich, no. A5316).

At the end of the in vitro NP exposures for 1, 6 or 24 hr, the cells were washed with PBS and lysed using NP40 cell lysis buffer (Invitrogen, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Sigma-Aldrich, USA). The cell lysates obtained were the subjected to SDS-PAGE followed by transfer onto a nitrocellulose membrane using the semi-dry transfer system (BioRad, USA). After blocking with 1% Superblock (Sigma-Aldrich, USA) blocking solution for 1 hr, the membranes were probed overnight with specific primary antibodies: Phospho p38(Thr180/Tyr182), p38 MAPK antibody, along with phospho-NFκB p65(Ser536) or NFκB p65 antibodies (Cell Signalling, USA), followed by washing with PBS. Proteins were detected using horseradish peroxidase-tagged secondary antibodies (Cell Signalling, USA) and enhanced chemiluminescence substrate (Pierce, USA), followed by exposure to photographic film (GE Healthcare, Australia).

Monocytes were lysed in NP-40 buffer and protein concentrations were determined using bicinchoninic acid (BCA) protein assay kit (Life Technologies, Paisley, UK) according to the manufacturer’s instructions. Proteins were boiled in Laemmli buffer at 95°C for 5 minutes before resolved by SDS–PAGE using 10% Tris-glycine gels and transferred onto methanol-activated Polyvinylidene difluoride membranes (Life Technologies) After transfer, membranes were blocked with 3–5% BSA in PBS with 0.1% Tween-20 (PBS-T; Fisher Scientific, Loughborough, UK) for 1 hour at room temperature, and incubated overnight at 4°C with the following primary antibodies diluted in 3–5% BSA in PBS-T; anti-SARM at 1:1000 (ProSci, Poway, USA), and anti-GAPDH at 1:3000 (Cell Signalling, London, UK). A secondary anti-rabbit HRP-conjugated antibody (Sigma-Aldrich, Gillingham, UK) was used for detection. For protein visualization, blots were incubated with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). Membranes were then removed from the ECL solution and exposed to photographic film (GE Healthcare, Little Chalfont, UK) and developed using a Konica Minolta SRX-101A film developer and Champion Photochemistry film developer and fixer solutions (Jet X-ray, London, UK).

Cells were plated in 10 cm diameter dishes. After 48 h, cell lysates were fractionated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare Life sciences). The membranes were blocked with 5% fat free milk and incubated with primary antibodies against Syntenin (homemade, see generation of syntenin antibody paragraph), β Actin (Santa Cruz, sc-69879), Tubulin (Sigma–Aldrich, USA), Cyclin D2 (Santa Cruz, sc-593), CDK4 (Cell signaling #2906), Retinoblastoma (BD Biosciences # 554136), and HRP-conjugated secondary antibodies. The membranes were washed with PBS/0.1% Tween buffer and antibody binding was revealed using enhanced chemiluminesescence (ECL) reagent (Thermo Scientific) according to the recommendations of the manufacturer. Signals were detected on photographic films (GE healthcare).