Gel imaging system

Cells were washed with PBS and lysed in ice-cold RIPA buffer (10 mM Tris-HCl, 1 mM EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1:100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, and 50 mM sodium fluoride). The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to detect the protein content of the lysates according to the manufacturer’s protocol. The proteins were loaded on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) and blocked with 5% nonfat milk powder in tris buffered saline tween (TBST). The membranes were incubated overnight with the following monoclonal primary antibodies: AIF (1:1000, rabbit, Abcam, USA), calpain (1:1000, mouse, Abcam, USA), active-caspase-3 (1:1000, rabbit, Abcam, USA), and β-actin (Santa Cruz Biotechnology, USA, 1:4000). The membranes were incubated with secondary conjugated horseradish peroxidase antibodies (Beyotime) for 2 h at room temperature. The blots were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA).The band intensities were obtained by a gel imaging system (Syngene, USA) and intensities were quantified with the software of image J.

In an initial screening 25 transformants of each construct were streaked on BMMY agar plates. Two clones from each construct with a high and low fluorescence were chosen as positive and negative controls respectively, and used as references in subsequent screenings of additional 96 Δ1-708 HaTRPA1 and 48 HaTRPA1 clones. The plates were incubated for 48 h at 30 °C, with 100 µL methanol in the lid, to induce protein expression as previously described [18 ]. The fluorescence was captured using a Gel imaging system (Syngene, Cambridge, UK), and evaluated by visual comparison.

Western blot analysis was performed as described [36 (link)]. Equal amounts of protein (15–20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane using Criterion XT Precast gels (Bio-Rad, USA). Membranes were blocked with 5% BSA for 2 h; then the corresponding antibodies GLUT4 (1 : 1000, #347063, Zen Bio), HK2 (1 : 1000, #200569, Zen Bio), PFKM (1 : 1000, #ab154804, Abcam), PKM (1 : 1000, #200667-1A7, Zen Bio), LDHA (1 : 500, #384822, Zen Bio), AMPKα (1 : 1000, #ab207442, Abcam), p-AMPKα (1 : 1000, #AP0116, ABclonal), mTOR (1 : 1000, #380411, Zen Bio), p-mTOR (1 : 1000, #5536, Cell signalling), 4EBP1 (1 : 500, #306002, Zen Bio), p-4EBP1 (1 : 500, #ab278686, Abcam), and Actin (1 : 2000, #200068-8F10, Zen Bio) were incubated overnight at 4°C; finally, blots were incubated with secondary antibodies at room temperature for 2 h. Bands were visualized with the gel imaging system (Syngene, UK) and analyzed with ImageJ analysis software (National Institutes of Health, https://rsb.info.nih.gov/ij/). The research met all the requirements of ARRIVE checklist and most of the requirements of CONSORT checklist if applicable (see supplemental files, named S1 ARRIVE Checklist and S2 CONSORT 2010 Checklist).

All CS-g-PEI/pDNA polyplexes were freshly prepared by a coacervation method which was widely used [41 (link),42 (link)]. Briefly, a solution of polymer was added to a solution of pDNA at specific N/P ratio under gentle vortex, and incubated for 30 min at room temperature before use. Unless specified otherwise, the N/P ratio of polymer to DNA in this paper is weight ratio.
To investigate the binding affinity of CS-g-PEI copolymers to pDNA, the CS-g-PEI/pDNA polyplexes with different N/P ratios were loaded on a 1% agarose gel with Tris-acetate (TAE) running buffer and subjected to electrophoresis at 90 V and 50 mA for 60 min (0.1 mg of pDNA per hole, the N/P ratio varying from 1 to 6). The fluorescence of the intercalated dye (ethidium bromide) was measured using a gel imaging system (Syngene, UK). To evaluate the stability of the polyplexes in serum, the freshly prepared polyplex (at the N/P ratio of 6) solutions and FBS solutions were mixed in Eppendorf tubes. After incubating at 37°C for 30 min, the stability of the polyplexes in serum was evaluated by electrophoresis.
Hydration particle size and ζ-potential of the polyplexes were measured in triplicate on a dynamic light scattering (DLS, Nano ZS 90, Malvern, UK) with 90° scattering angles at 25°C. The CS-g-PEI/pDNA polyplexes were prepared in water at the N/P ratio of 6.

Cells were collected and lysed in lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). Lysates were clarified by centrifugation at 4°C for 15 min at 13,000 ×g and the concentration of protein in the supernatant was measured using a bicinchoninic acid (BCA) assay kit with a Varioskan multimode microplate spectrophotometer (Thermo Waltham, MA). Subsequently, proteins (30 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 5% nonfat milk for 1 h, incubated with primary antibodies (listed above) for 1 h at 37°C and overnight at 4°C, rinsed in PBST, and finally incubated in secondary antibodies for 1 h at room temperature. Blots were visualized by using an enhanced chemiluminescence kit (Millipore, Billerica, MA). Digital images of blots were produced by a Syngene Gel Imaging System and quantified with GeneSnap (Syngene) software.