Ab184601

The CD68 plasmid, which simultaneously expresses EGFP under the control of the CD68 promoter, was used to determine the plasmid transfection efficiency of the synthetic DNA nanocarriers, and the CMV plasmid with the CMV promoter was used as a control. Briefly, RAW264.7 cells (2 × 10⁵ cells/well) were seeded in six-well plates for 24 h, and the cells were then incubated with synthetic DNA nanocarriers with different structural integrities (including free plasmid, P/PB nanocarriers containing plasmid DNA and PBAE, P/PB/N nanocarriers containing plasmid DNA and PBAE C32-122-NLS, P/PB/N/R nanocarriers containing plasmid DNA, PBAE C32-122-NLS and PGA–peptide) at plasmid doses of 50 nM. After incubation for 6 h, the medium was replaced with fresh medium, and the cells were further incubated for 48 h. Afterwards, the percentage of EGFP-positive cells was analyzed by a flow cytometry assay (FACS Calibur flow cytometer, BD Biosciences, USA), and after further incubation for 48 h, EGFP expression was analyzed by western blotting. A monoclonal antibody against EGFP (Abcam, cat. no. ab184601) was used at a dilution of 1:1000, and goat anti-mouse IgG-HRP (1:5000, Abcam, cat. no. ab6789) was used as the secondary antibody. The results were visualized using an ImageQuant LAS 4000 mini system (GE Healthcare, UK).

A total of 0.5 g silk was added to 2 mL 9.3 M LiBr solution and completely dissolved at 60 °C for 4–6 h. The total protein concentration was measured with a BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). A 100 μg mass of total protein was electrophoresed by 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking at 25 °C for 2 h, the primary antibodies, including rabbit anti-SER3 and rabbit anti-P25 (synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd.), mouse anti-EGFP antibody (ab184601, abcam, UK) and mouse anti-beta-Tubulin (ab108342, abcam, UK) was added to the membranes and incubated at 4 °C for 12 h, respectively. The membranes were washed three times with TBST. HRP-labeled goat anti-rabbit IgG or HRP-labeled goat anti-mouse IgG (Bioworld Technology, Minneapolis, MN, USA) was added and incubated at 37 °C for 2 h. Under dark conditions, 1 mL EZ-ECL chemiluminescence reagent was added to the membrane, and the bands were observed through chemiluminescence detection (1708370, Bio-Rad, USA) after 1 min. The images were analyzed with Image Lab (Bio-Rad, USA). The dilution of primary antibodies was 1:2000, and the dilution of secondary antibodies were 1:5000. All original blots are shown in source data.

Whole-cell protein extracts were prepared using RIPA lysis buffer (PC101, EpiZyme, China). Proteins were separated on a 10% SDS/PAGE gel and electroblotted on to an Immobilon-P membrane (Millipore). The membrane was blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline containing Tween 20 (TBST) buffer (20 mM Tris/HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20), then incubated with the primary antibody overnight at 4°C, washed with TBST buffer, and incubated with HRP-conjugated secondary antibody for 1.5 h at room temperature. After being washed with TBST buffer, blots were developed with SuperSignal West Pico or Femto chemiluminescent substrate (Pierce) or ECL Plus Western blotting detection reagents (GE Healthcare). Antibodies used were rabbit monoclonal to α-tubulin (ab179484, Abcam, Cambridge, U.K.), rabbit monoclonal to EGFP (ab184601, Abcam, Cambridge, U.K.). Proteins recognized by the antibodies were detected by ImageQuant LAS 500 (GE, CT, U.S.A.) using HRP-conjugated Goat Anti-Rabbit secondary antibody (BBI, Sangon Biotech, China).

Egg white from different chickens was collected and 1:1000 diluted into PBS and their concentration was detected by bicinchoninic acid (BCA) assay reagent (Beyotime Biotechnology). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer was then mixed with samples and boiled for 5 min and equal amounts of proteins (20-40 μg) were electrophoresed on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). For first antibodies incubation, the monoclonal antibody against EGFP (Abcam, ab184601) and Ovalbumin (Abcam, ab306591) was used, and PVDF membranes were overnight incubated in 4 °C with 1st antibodies. 2nd antibodies labeled with alkaline ahosphatase (Beyotime) and BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime) were used for visualization of EGFP protein or ovalbumin protein bands. The band grey density assays were performed by using ImageJ.

Mice were anaesthetized and transcardially perfused at either 3, 8 or 12 months of age with PBS. Brains were removed and the hemispheres separated. One hemisphere of the brain was immersion‐fixed in 4% paraformaldehyde for immunohistochemical analysis as previously described [22 (link), 23 (link)], and the other hemisphere was sub‐dissected and snap‐frozen in liquid nitrogen for biochemical analysis. Fixed brains were processed using an automated system (Excelsior, Thermo, USA), embedded in paraffin and sagittally sectioned at the level of the mid‐hippocampus into 3‐μm‐thick sections using a microtome (Thermo, USA). All staining was done in Sequenza staining racks for standardisation using previously reported protocols [24 (link)]. The following primary antibodies were used: pan‐TDP‐43 (10782‐2‐AP, Proteintech), POU3F2 (ab94977, Abcam), GFAP (G9269, Sigma), EGFP (ab184601, Abcam), Cyclin F (sc‐515207, Santa Cruz) and VCP (sc‐20799, Santa Cruz). Microscopy was performed with an Olympus BX51 (United States) epi‐fluorescence microscope equipped with an XM10 MONO camera, or sections were scanned with an Axio Scan Z1 automated slide scanner (Zeiss).