Cytc antibody

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.