iPSC lysate was prepared in RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% IGEPAL CA-630, 1 mM phenymethylsulfonyl fluoride (PMSF), 20 mg/ml pepstatin A, 20 mg/ml leupeptin, 20 mg/ml aprotinin, 50 mM NaF and 1 mM Na3VO4). The lysate was resolved by 10% Bis–Tris NuPAGE (ThermoFisher Scientific). The proteins resolved by SDS–PAGE were transferred to the Nitrocellulose (NC) membrane (Satorious) and western blot analysis was conducted by using the following antibodies: mouse anti-MAPK3 (12D11, cat# MA1-13041, ThermoFisher Scientific), rabbit anti-β actin (cat# ab8227, Abcam)43 (link). The secondary antibodies used were HRP-conjugated goat-anti mouse IgG antibody (Abcam) and HRP-conjugated goat-anti rabbit IgG antibody (Abcam). Membrane was developed by reacting with chemiluminescence HRP substrate (Millipore) and exposed to the Amersham Hyperfilm ECL (GE Healthcare) for visualization of protein bands. The protein bands were quantified by using the NIH Image J Software. Statistical Analysis for western blot was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s adjustment for multiple comparisons.
Asupplementary methods checklist is available.
A