Ab52761

Elution fractions containing CaN were collected as described above in ‘Purification and quantification of CaM binding partners’. In preparation for SDS-PAGE analysis, Laemmli buffer containing 5% 2-mercaptoethanol was added to each protein-containing elution fraction at a 1:3 ratio. Fractions were boiled at 95°C for 5 minutes and were loaded equally by volume into a 4–15% gradient gel (PROTEAN®TGX™ Precast Gels, Bio-Rad). Proteins were transferred to a 0.2 μm PVDF membrane (ThermoFisher Scientific, St. Louis, MO) and probed with primary (Abcam ab52761 for α-subunit and ab49658 for β-subunit) and secondary antibodies (IRDye® 800CW and 680RD, LI-COR), then analyzed with Odyssey CLx imaging system (LI-COR).

The western blotting assay was performed by well-established protocols as previously described (19 (link)). Primary antibodies used in this study were anti-ABL1 antibody (1:500, ab85947, Abcam, Cambridge, MA), anti-Bcl-2 antibody (1:300, BCL/10C4, Biolegend, San Diego, CA), Anti-Bcl-xl antibody (1:500, sc-136207, Santa Cruz Biotechnology, Dallas, TX), anti-Bax antibody (1:300, 2D2, Biolegend), anti-β-actin antibody (1:500, 2F1-1, Biolegend), anti-GAPDH antibody (1:500, FF26A/F9, Biolegend), anti-p27 antibody (1:300, sc-56338, Santa Cruz Biotechnology), anti-cyclin-D1 antibody (1:500, sc-8396, Santa Cruz Biotechnology), anti-IRS1 antibody (1:500, ab52167, Abcam), anti-AKT2 antibody (1:500, ab175354, Abcam), anti-PPP3CA antibody (1:10000, ab52761, Abcam), anti-TGFβ1 antibody (1:100, ab92486, Abcam), anti-MAP2K2 antibody (1:500, sc-81473, Santa Cruz Biotechnology), anti-PI3K-p11a antibody (1:1000, ab151549, Abcam). Secondary antibodies used were: anti-mouse IgG HRP-conjugated secondary antibody (1:5000, sc-516102, Santa Cruz Biotechnology), and anti-rabbit IgG HRP-conjugated secondary antibody (1:5000, sc-2357, Santa Cruz Biotechnology).