Odyssey image analysis system

Whole cell extracts (WCE) were prepared and immunoblotting analysis performed using the Odyssey Image Analysis System (Li-Cor Biosciences, Cambridge, UK), as previously described [19 (link),20 (link)]. Primary antibodies raised against ASAH1 (1:500) (BD Biosciences, Wokingham, UK), poly-ADP ribose polymerase-1 (PARP-1) (1:5000) (Santa Cruz Biotechnology, Heidelberg, Germany) or actin/tubulin (1:20,000) (Sigma-Aldrich, Gillingham, UK), along with Alexa Fluor 680 or IR Dye 800 secondary antibodies (1:20,000) (Li-Cor Biosciences, Cambridge, UK) were used for detection.

Cells were irradiated with 10 Gy X rays, α-IR, or protons or treated with 150 μM H₂O₂ for 15 minutes and harvested by centrifugation (1500 rpm, 5 minutes, 4°C). To inhibit DNA transcription, cells were preincubated for 1 hour with 1 μg/mL actinomycin D before irradiation. Histones were purified by acid extraction, as previously described (21) (link), and analyzed by quantitative Western blotting using the Odyssey image analysis system (Li-cor Biosciences, Cambridge, United Kingdom) as described in the Supplementary Information (available online at www.redjournal.org) from at least 3 independent experiments.

To analyze DNA glycosylase cleavage activity, hNEIL1 and hNEIL3^FL, diluted in 25 mM HEPES pH 7.9, 100 mM KCl, 12 mM MgCl₂, 1 mM EDTA, 17% glycerol and 2 mM DTT, were incubated with 5 nM of oligonucleotide substrate molecules for 30 min at 30 °C in reaction buffer (40 mM HEPES pH 7.8, 5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM DTT, 2 mM ATP, 0.1 mg/mL BSA) in a final volume of 10 µL. This is a standard reaction buffer used in our laboratory for BER reactions, although ATP and MgCl₂ are not required for DNA glycosylase activity. Reactions were stopped by the addition of an equal volume of formamide loading dye (95% formamide, 0.05% bromophenol blue) and heated to 95 °C for 5 min. Reaction products were separated by 10% denaturing-PAGE and subsequently quantified using the Odyssey Image Analysis System (Li-Cor Biosciences, Cambridge, UK). For sodium borohydride trapping of hNEIL3, reactions were supplemented with 100 mM sodium borohydride and the reaction performed at 30 °C for 30 min. The reaction was stopped by the addition of 10 µL of 3× SDS-PAGE sample buffer (25 mM Tris-HCl pH6.8, 2.5% 2-mercaptoethanol, 1% SDS, 10% glycerol, 1 mM EDTA and 0.05% bromophenol blue) and heated to 95 °C for 5 min. Samples were separated by 10% Tris-Glycine SDS-PAGE and the gel was visualized using the Odyssey Imaging Analysis System.

Whole cell extracts for Western blotting were prepared as described previously (16 (link)). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon-FL Polyvinylidene fluoride (PVDF) membranes (Millipore) according to standard procedures (Novex). Blots were probed with following antibodies: XRCC1 (Neomarkers, MS-1393-P0), DNA polymerase β (raised in-house and affinity-purified), α-tubulin (Sigma, T6199), MCM4 (abcam, ab4459-50), RP-A p32 (Bethyl, A300-244A), PCNA (Santa Cruz, sc-56), α-actin (Abcam, ab6276), PARP-1 (raised in-house and affinity-purified), p21 (Cell signaling, 12D1), PSAT1 (Novusbio, 21020002), PHGDH (Sigma, HPA021241), p53 (Santa Cruz, sc-126), PNKP (Abnova, H00011284-B01). Secondary antibodies conjugated with Alexa Fluor 680 (Molecular Probes) and IRDye^® 800 (Rockland) fluorescent dyes were used. Detection and quantification was carried out using an Odyssey image analysis system (Li-Cor Biosciences).

Whole cell extracts for Western blotting were prepared as described previously [57] (link). Proteins were separated on 4% to 20% Tris-Glycine gels (Novex) and transferred onto Immobilon-FL Polyvinylidene fluoride (PVDF) membranes (Millipore) according to standard procedures (Novex). Blots were probed with following antibodies: CDK6 (Novusbio, NBP1–87,262), CDK4 (Santa Cruz, sc23896), Cyclin D1 (Santa Cruz, sc8396), and α-Tubulin (Sigma, T5168). Secondary antibodies conjugated with Alexa Fluor 680 (Thermo Scientific) and IRDye 800CW (Li-cor Biosciences) fluorescent dyes were used. Detection and quantification were carried out using an Odyssey image analysis system (Li-cor Biosciences).

Western blot analysis was performed as previously described in our studies [21 (link), 24 (link)]. Lung tissues were dissected and lysed using iced-cold lysis buffer (Beyotime, catalog no. P0013G). Protein concentration was determined via standard BCA assay. After gel electrophoresis, protein was then transferred to a nitrocellulose membrane and blocked using 5% nonfat milk for 1 h. Membranes were incubated with rabbit anti-mouse WISP1 antibody (1:400, R&D systems) and β-actin antibody (1:10000, Proteintech) at 4 °C overnight, and then incubated with secondary antibody for 1 h at 37 °C. Odyssey image analysis system (Licor Biosciences) was used to quantify.

AMΦ and lung tissues were lysated in radio-immunoprecipitation lysis buffer (RIPA), protease inhibitors (Roche, Mannheim, Germany) and phenylmethylsulfonyl fluoride (PMSF). Protein concentrations were subsequently determined by standard BCA assay. After addition of 6 × sodium dodecyl sulfate (SDS) loading buffer, equivalent amounts of protein were heated (100 °C; 5 min) and separated by gel electrophoresis using a 10% SDS-polyacrylamide electrophoresis gel. Resolved proteins were then transferred to a nitrocellulose membrane and blocked with Tris-buffered saline containing Tween-20 (TBST) and 5% nonfat milk (1 h; 24 °C). Nitrocellulose membranes were incubated overnight at 4 °C with primary antibody against TLR3 (ab62566; Abcam, Hong Kong, China), NF-κB p65 (ab7970, Abcam, Hong Kong, China), Abcam, MIP-2 (ab25130, Hong Kong, China), PCNA (ab18197, Abcam, Hong Kong, China) and β-actin (ab8226, Abcam, Hong Kong, China). The membranes were washed in TBST three times, incubated with secondary antibody (926-32221 IRDye 680 mouse-anti-rabbit secondary antibody; Licor Biosciences, Lincoln, NE, USA) for 1 h at 37 °C and then washed in TBST three additional times. The membranes were determined by using an Odyssey image analysis system (Licor Biosciences). Western blots were quantitated using Quantity One software (Bio-Rad, Foster City, CA, USA) and normalized to β-actin and PCNA signal.