Immunoblotting Protocol for Protein Analysis

For immunoblot analysis, cells were washed twice with PBS and lysed in lysis buffer (1% NP-40, 120 mM NaCl, 40 mM Tris-HCl (pH7.4), 1.5 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and protease inhibitor cocktail (Roche)), as described in (Cai et al. 2020 (link); Lee et al. 2020 (link)). Primary antibodies against phospho-Akt (9272), Akt (4056), phospho-ERK (9101), ERK (4696) (Cell Signaling), α-tubulin (T5169; Sigma), and horseradish peroxidase-conjugated secondary antibodies were used. IPMK antibody was generated with a peptide targeting mouse IPMK amino acids 295-311 (SKAYSRHRKLYAKKHQS) from Covance.