For total protein extraction, Arabidopsis seedlings were grounded in the presence of liquid nitrogen to fine powder, and total protein was extracted with 2 × SDS sample buffer (100 mM Tris-Cl, pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and freshly added 10% β-mercaptoethanol). Aliquots of denatured total protein were separated by SDS-PAGE and transferred to a PVDF membrane. For the detection of phosphorylated CBL proteins, total protein was separated by 10% SDS-PAGE with 15 μM Phos-tag (AAL-107, WAKO pure chemical industries, Ltd) and transferred to PVDF membrane. For immunoblot analyses, anti-CBL3 (19 (link), 66 (link)), anti-GAPDH (PHYTOAB, PHY0303A), anti-actin (PHYTOAB, PHY0001), anti-Flag (Sigma-Aldrich, A8592-2MG), anti-S6K1-p (phosphor T449) (Abcam, ab207399), anti-HA (Santa Cruz Biotechnology, sc-7392HRP) were used as primary antibodies. Each experiment was repeated at least three times, and one representative result is shown in the figures. Band density in immunoblots was quantified using Image J software.
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Arabidopsis Protein Extraction and Detection
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Variable analysis
independent variables
- Treatment with 15 μM Phos-tag in SDS-PAGE
- Phosphorylation status of CBL proteins
- Protein expression levels of CBL3, GAPDH, actin, S6K1-p (phosphor T449)
- Arabidopsis seedlings
- Liquid nitrogen grinding
- 2× SDS sample buffer (100 mM Tris-Cl, pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and freshly added 10% β-mercaptoethanol) for protein extraction
- SDS-PAGE separation of denatured total protein
- Transfer to PVDF membrane
- Primary antibodies: anti-CBL3, anti-GAPDH, anti-actin, anti-Flag, anti-S6K1-p (phosphor T449), anti-HA
- Not explicitly mentioned
- Not explicitly mentioned
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