Cells (1X106 cells) were lysed in RIPA buffer (Beyotime, Beijing, China), and the protein samples were segregated using SDS-PAGE. The samples were transferred onto nitrocellulose membranes, and the membranes were blocked in 5% skim milk. Membranes were incubated with primary antibodies: anti-WDR62 and anti-β-actin (1:2000), anti-p-DNA-PK (DNA-dependent protein kinase) and anti-Rad51 (1:2500), anti-p-ERK (extracellular signal-regulated kinase) and anti-ERK (1:3000), anti-p-JNK and anti-JNK (c-Jun N-terminal kinase) (1:3500), anti-p-p38 and anti-p38 (1:4000). The membranes were then incubated with secondary antibodies (1:4500), and subjected to chemiluminescence reagent kit (Beyotime) according to previous study [15 (link)]. All the antibodies were purchased from Abcam.
Protocol detail
Western Blot Analysis of Cellular Signaling Pathways
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Actin
Antibodies
Buffer
C jun n terminal kinase 1
Cells
Chemiluminescence
Dna dependent protein kinase
Erk 1
Extracellular signal regulated kinase
Membranes
Milk
Nitrocellulose
Protein
Ripa
Sds page
Corresponding Organization : Ningbo University
Other organizations : Ningbo No.6 Hospital
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Variable analysis
independent variables
- Cell lysis in RIPA buffer
- SDS-PAGE for protein separation
- Transfer of samples onto nitrocellulose membranes
- Blocking of membranes in 5% skim milk
- Incubation with primary antibodies: anti-WDR62 and anti-β-actin (1:2000), anti-p-DNA-PK and anti-Rad51 (1:2500), anti-p-ERK and anti-ERK (1:3000), anti-p-JNK and anti-JNK (1:3500), anti-p-p38 and anti-p38 (1:4000)
- Incubation with secondary antibodies (1:4500)
- Chemiluminescence reagent kit (Beyotime)
- Protein expression levels of WDR62, β-actin, p-DNA-PK, Rad51, p-ERK, ERK, p-JNK, JNK, p-p38, and p38
- Cell count (1X10^6 cells)
- RIPA buffer (Beyotime, Beijing, China)
- Nitrocellulose membranes
- Antibodies (purchased from Abcam)
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