Western blot analysis was conducted as described previously [18 (link)]. Briefly, PCa cells were lysed in RIPA buffer (#KGP250, KeyGEN) following the manufacturer’s instructions. Then, 25 µg of protein was separated by SDS‒PAGE and transferred to PVDF membranes (Millipore). Subsequently, membranes were blocked with 5% nonfat milk. After washing, the primary antibodies were immunoblotted. The following primary antibodies were used in this research: DDX17 (ab180190) was purchased from Abcam, and TGIF2 (sc -81,989) and GAPDH (sc -365,062) were purchased from Santa Cruz. After incubation overnight, HRP-conjugated anti-mouse (sc-2005) and anti-rabbit IgG (sc-2004) secondary antibodies (Santa Cruz) were added. Finally, the signals were visualized by Bio-Rad ChemiDoc XRS+.
Protocol detail
Western Blot Analysis of PCa Cell Lysates
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Anti igg
Antibodies
Buffer
Cells
Gapdh
Membranes
Milk
Mouse
Protein
Pvdf
Rabbit
Ripa
Sds page
Western blot analysis
Corresponding Organization : Third Affiliated Hospital of Guangzhou Medical University
Other organizations : First Affiliated Hospital of Guangzhou Medical University, Huizhou Central People's Hospital
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Variable analysis
independent variables
- Not explicitly mentioned
- Protein expression levels of DDX17, TGIF2, and GAPDH
- Protein concentration used for SDS-PAGE (25 µg)
- Blocking buffer (5% nonfat milk)
- Primary antibodies (DDX17, TGIF2, GAPDH)
- Secondary antibodies (HRP-conjugated anti-mouse and anti-rabbit IgG)
- Positive control: Not specified
- Negative control: Not specified
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