Anti p44 42 mapk erk1 2 antibody

Around 200 mg of mycelia was harvested and ground into powder with liquid nitrogen, and resuspended in 1 ml of extraction buffer (10 mM Tris‐HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X‐100) with 1 mM phenylmethanesulfonyl fluoride (PMSF) and 10 μl of protease inhibitor cocktail (Sigma). Total proteins were separated by 12% SDS‐PAGE and transferred to nitrocellulose membranes. The Pmk1 protein level was assayed by using anti‐p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), and the phosphorylated Pmk1 was assayed by anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell Signaling Technology).

Podocytes were lysed in a SDS sample buffer containing 0.5 M Tris-HCl, 10% SDS, glycerol, bromophenol blue, and 3% 2-mercaptoethanol. They were boiled at 95°C for 10 min, and then the protein was fractionated on 10%–15% polyacrylamide gels (e-PAGEL, ATTO Corporation, Japan). The protein was transferred to PVDF membranes (Immobilon-P Transfer Membrane); the membranes were blocked for 1 h at room temperature to block the nonspecific binding of the protein and incubated for 18 h at 4°C with primary antibodies. The primary antibodies that were used were as follows: anti-VPAC1 antibody (1 : 200 dilution, Santa Cruz: sc-30019), anti-phospho-p44/42 MAPK (extracellular signal-regulated kinase (ERK1/2)) antibody (1 : 1000 dilution, Cell-Signaling: number 9106), and anti-p44/42 MAPK (ERK1/2) antibody (1 : 1000 dilution, Cell-Signaling: number 9102). The blots were then washed and incubated with second antibodies, peroxidase-conjugated anti-rabbit immunoglobulins (1 : 2500 dilution, GE healthcare), or goat anti-mouse IgG-HRP (1 : 2500 dilution, Santa Cruz: sc-2055) for 1 h at room temperature. After washing for several times, the antibody binding sites were visualized using an ECL western blotting detection system (GE healthcare: RPN2106). The blots were quantified using a ChemiDoc MP ImageLab PCsystem (BIO-RAD).

3T3L1 cells (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and they were transfected with Silencer select Pre-designed siRNA Lgals9 (Gal-9 siRNA) and silencer select negative control siRNA (NC siRNA) (Thermo Fisher Scientific, Cat#s69189) by Lipofectamine RNAi MAX (Cat#13,778,030). After 40 h, the 3T3L1 cells were further cultured for 20 minutes in the absence and presence of 10 μM H₂O₂.
The cells were lysed in 500 μl of RIPA buffer and subject to SDS-PAGE under non-reducing conditions and Western blot analysis using anti-galectin-9 antibody (Biolegend, Cat#137,902), anti-peroxiredoxin 2 (PRDX2) antibody (abcam, Cat#ab109367), anti-GAPDH antibody (Cell Signaling Technology, Cat#8884), anti-phospho-p38 MAPK (Thr180/Try182) antibody (Cat#9211), anti-p38 MAPK antibody (Cat#9212), anti-phospho-SAPK/JNK (Thr183/Try185) antibody (Cat#9251), anti-SAPK/JNK antibody (Cat#9252), anti-phospho-p44/42 MAPK (Erk1/2)(Thr202/Try204) antibody (Cat#9101), and anti-p44/42 MAPK (Erk1/2) antibody (Cat#9101).