Fig. 3.

Phosphatase inhibitors are critical to preserve certain phosphorylated residues under certain lysis conditions. (A, B) Effect of lysis buffer and presence or absence of phosphatase inhibitors (PPIs) on the detection of phosphorylated Akt (p-Akt) on Thr³⁰⁸ (T308) and Ser⁴⁷³ (S473). (C) Effect of lysis buffer and presence or absence of PPIs on detection of glycogen synthase kinase-3α/β phosphorylated on Ser21 and Ser9 (p-GSK3α/β). (D) Effect of lysis buffer and presence or absence of PPIs on detection of glycogen synthase phosphorylated on Ser⁶⁴¹ (p-GS). AC16 cells were lysed in RIPA or NP-40 lysis buffer with or without PPIs. Vinculin, tubulin, GAPDH, and actin were used as loading controls where indicated. Total Akt, GSK3α/β, and GS were used to monitor specific changes in protein abundance. Band intensities were quantified from the 16-bit digital image by densitometry in ImageJ and are shown normalized to the average +PPI conditions for each target across both lysis conditions. Data are representative of two experiments.