Anti dhx15 sc 271686

After fixation, permeabilization and blocking, the cells were incubated with primary antibodies and followed by incubation with secondary antibodies. Nucleus were stained with DAPI. Images were captured under a fluorescence microscope (Leica, Germany). Antibodies applied in IF assay were as follows: anti-DHX15 (sc-271686, Santa Cruz Biotechnology, USA), anti-NF-κB p65 (sc-8008, Santa Cruz Biotechnology, USA), Alexa Fluor 488-goat anti-mouse IgG(H + L) (YEASEN, Shanghai, China), and Cy3-goat anti-mouse IgG(H + L) (YEASEN, Shanghai, China).

Four-week-old female BALB/c nude mice purchased from SLAC (Shanghai, China) were divided into two groups randomly (n = 5 for each group). A total of 2 × 10⁶ MDA-MB-231 cells stably expressing circRNF10 or vector were injected subcutaneously. All mice were sacrificed after 6 weeks and tumors were collected. The weight and volume of tumors were determined. For IHC assay, tumors were fixed, dehydrated, embedded, and sliced. The sections were then stained with anti-Ki-67 (GB121142, Servicebio, Wuhan, China), anti-DHX15 (sc-271686, Santa Cruz Biotechnology, USA), and anti-NF-κB p65 (sc-8008, Santa Cruz Biotechnology, USA). Representative images were taken using Leica Microsystems (Germany).

The probe of circRNF10 for RNA pulldown was as follows: 5’-GGCUACAAAUGCGCUCCUAGAUGAA-3’ (GenePharma, Shanghai, China). Proteins binding to circRNF10 were pulled down using RNA pulldown Kit (BersinBio, Guangzhou, China) and separated by SDS-PAGE electrophoresis. Silver staining was then conducted using Protein Silver Stain Kit (YEASEN, Shanghai, China). The differential protein band was identified by mass spectrometry (IBSbio, Shanghai, China). RIP was conducted using RNA Immunoprecipitation Kit (BersinBio, Guangzhou, China), followed by RT-PCR and agarose gel electrophoresis. Anti-DHX15 (sc-271686, Santa Cruz Biotechnology, USA) and anti-GFP (sc-9996, Santa Cruz Biotechnology, USA) were used for RIP.