Tdp 43 antibody

Samples were prepared with Tris-glycine SDS buffer (Novex, Life Technologies) containing 10% β-ME at a 1:1 ratio (v/v) and heated at 95°C for 5 min. Equal amounts of protein were loaded into 10-well 4–20% Tris-glycine gels (Novex). After transfer, blots were blocked with 5% nonfat dry milk in TBST for 1 h and then incubated with rabbit anti-sera (1:5000), B cell supernatant (1:500), mAbs (26H10, 2E9 and 23A1; 1:500), rabbit polyclonal GFP antibody (1:1000, Thermo Fisher Scientific, A-11122), rabbit polyclonal TDP-43 antibody (1:1000, Proteintech, 12892-1-AP) or mouse monoclonal GAPDH antibody (1:5000, Meridian Bioscience, H86504M) overnight at 4°C with rocking. Membranes were then washed in TBST three times for 10 min each and incubated for 1 h with donkey anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (1:5000, Jackson ImmunoResearch) for 1 h at room temperature. Protein expression was visualized by enhanced chemiluminescence treatment using Western Lightning Plus-ECL (Perkin Elmer).

Reagent-grade ICT, (purity 99.5% by HPLC analysis) was obtained from Zhongke Quality Inspection Biotechnology Co., Ltd. (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the medium was less than 0.1% (v/v). Human TDP-43 and polybrene were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China), a TransZol Up Plus RNA Kit and EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix were purchased from TransGen Biotechnology Co., Ltd. (Beijing, China) and NuHi Robustic SYBR Green Mix was purchased from Nuhigh Biotechnologies Co., Ltd. (Suzhou, China). High-Sig ECL Western Blotting Substrate was purchased from Tanon Technology Co., Ltd. (Shanghai, China). RIPA buffer (high) (R0010) was purchased from Solarbio Life Science (Beijing, China). A GAPDH antibody, a TDP-43 antibody, a CytC antibody, and HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) were purchased from Proteintech Group (Wuhan, China). A goat anti-mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific (Waltham, USA).

Cells were collected, lysed with RIPA lysis buffer, and then centrifuged at 12,000 rpm at 4 °C for 15 min. The total protein concentration was measured with a BCA protein assay kit (Beijing Solarbio). The proteins were then heated at 100  °C for 5 min for denaturation. Equal amounts of total protein (25 µg per lane) were loaded on 12% SDS-PAGE gels and separated. After sample loading, the voltage was set to 90 V and then to 120 V until the sample reached the bottom of the gel. The proteins were then transferred to a nitrocellulose (NC) film by the sandwich method. The membrane was washed three times with TBST and then blocked with 5% skimmed milk. The membrane was then incubated for 12 h at 4 °C with a GAPDH antibody (1:1000; Proteintech Group), TDP-43 antibody (1:1000; Proteintech Group), and CytC antibody (1:1000; Proteintech Group). The membrane was washed with TBST 3 times and incubated with HRP-conjugated Affinipure goat anti-rabbit lgG (H+L) (1:1000; Proteintech Group) for 2 h at room temperature. The membranes were developed using hydrogen peroxide and Supersignal West Pico Luminol (Pierce, Seymour Fisher Technologies). Finally, High-Sig ECL Western Blotting Substrate (Shanghai Tanon Technology Co., Ltd.) was used to visualize the membrane.

For each CUT&RUN reaction, 5×10⁴ cells were used. CUT&RUN was performed using the CUT&RUN assay kit following manufacturer’s instruction (Cell Signaling Technology). Briefly, cells were dissociated with accutase. The cell suspension was centrifuged at 600 × g for 3 min at room temperature and the supernatant was removed. Cell pellets were washed 3 times with 1x wash buffer. Then cell pellets were resuspended in Digitonin buffer (prepared using the material from the kit) and incubated with primary antibodies and activated Concanavalin A Magnetic Beads at 4°C for overnight. After washing with Digitonin buffer once, bound materials were treated with pAG-MNase at 4°C for 1 h. Then the pAG-MNase was activated by adding Calcium Chloride to samples and incubated with samples at 4°C for 30 min. To stop the reaction, the stop buffer with spike-in DNA prepared from the kit was added to samples and incubated with samples at 37°C for 10 min. DNA on bound materials were collected and purified using spin columns. Samples were quantified using qPCR. Bound DNA was normalized with spike-in DNA and presented as the percentage of input DNA. TDP-43 antibody (Proteintech, 5μg per reaction) was used for the CUT&RUN assay. The primers used for CUT&RUN-qPCR of the CLU SNP1 include forward primer 5′-GGC TGC AGA CTC CCT GAA TC-3′ and reverse primer 5′-GCA AGG GCC CGT TAG AGA AT-3′.

Immunohistochemistry of FTLD-TDP superior frontal cortex was carried out as previously described (Phan et al., 2021 (link)). Briefly, formalin-fixed, paraffin-embedded sections (10 μm) were deparaffinized in xylene and rehydrated through graded ethanol, followed by antigen retrieval with citrate buffer (pH 6.0) using a pressure cooker (Aptum Bio Retriever 2,100, Aptum Biologics Ltd., United Kingdom) at a peak temperature of ~121°C and gradually cooling to room temperature. Endogenous peroxidase was blocked with 1% hydrogen peroxide in 50% ethanol. Sections were probed with TDP-43 antibody (Proteintech, 10,782-2-AP, 1:400) and NeuN antibody (Biolegend, SIG-39860, 1:100), washed with PBS and incubated with the corresponding secondary antibodies (Thermo Fisher Scientific, A-10042 and A-31571, 1:250) and 4′,6-diamidino-2-phenylindole DAPI (Sigma-Aldrich, D9542, 1 mg/ml). The slides were treated with 70% Sudan Black for 30 min and 10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5.0) to quench auto-fluorescence signals prior to cover-slipping with anti-fade fluorescence mounting medium (DAKO, S3023) and then sealed with nail polish. Negative controls (without primary antibodies or secondary antibodies) were performed for each immunohistochemistry run, and no signals were detected in each case.