Anti phospho p44 42 mapk polyclonal antibody

The full-length of GST-MKK10.2 and MBP-MPK6 fusion proteins were expressed and purified via E. coli strain BL21 (DE3) by standard protocols using amylose agarose beads and glutathione magnetic beads (Solarbio), respectively. For in vitro kinase assay, fusion proteins were incubated with 25 mM Tris-HCl (pH 7.4), 12 mM MgCl₂, 1 mM DTT, and 1 mM ATP at 30 °C for 45 min and stopped by SDS loading buffer. MPK6 phosphorylation level was evaluated using anti-phospho-p44/42 MAPK polyclonal antibody (Cell Signaling Technology, #9101, 1:2000 dilution). Loading controls of GST-MKK10.2, MBP-MPK6 were detected using anti-GST (Beijing Protein Innovation, AbM59001-2H5-PU, 1:5000 dilution), anti-MAPK6 (Beijing Protein Innovation, AbP80140-A-SE, 1:2000 dilution), respectively.

Preparation of protein extracts and Western blot analysis were performed as described previously19 (link). Anti-GFP monoclonal antibody JL-8 (Clontech), anti-phospho-p38 MAPK monoclonal antibody D3F9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-phospho-p44/42 MAPK polyclonal antibody (Cell Signaling), anti-Mpk1 polyclonal antibody yN-19 (Santa Cruz), and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Westren (Merck Millipore). Signal intensities were quantified by ImageQuant (GE Healthcare), and statistical analysis was performed with Excel (Microsoft).

Preparation of protein extracts and Western blot analysis were performed as described previously^{9 (link)}. Anti-phospho-p38 MAPK monoclonal antibody D3F9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-phospho-p44/42 MAPK polyclonal antibody (Cell Signaling), anti-Mpk1 polyclonal antibody yN-19 (Santa Cruz), anti-Myc monoclonal antibody 9E10 (Santa Cruz) and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore) or the Odyssey Imaging Systems (LI-COR Biosciences). Signal intensities were quantified by the Odyssey Imaging Systems, and statistical analysis was performed with Excel (Microsoft).