Mouse anti akt pan

Antibodies used for immunoblotting were: rabbit anti-pErbB2(Y1221/1222) mAb, rabbit anti-pErbB3(Y1289) mAb, rabbit anti-pAkt(T308) mAb, rabbit anti-pAkt(S473) mAb, rabbit anti-pERK1/2(T202/Y204) pAb, mouse anti-ERK1/2 mAb, mouse anti-Akt(pan) mAb, rabbit anti-PI3K p110α mAb (Cell Signaling, Danvers, MA, USA), mouse anti-ErbB2 mAb, mouse anti-ErbB3 mAb (Thermo Scientific, Fremont, MA, USA), and mouse anti-tubulin mAb (Sigma-Aldrich, St Louis, MO, USA). HRP-labeled secondary anti-mouse and anti-rabbit IgG antibodies were from GE Healthcare (Buckinghamshire, UK) and IRDye-680LT anti-rabbit IgG or IRDye-800CW anti-mouse IgG conjugated secondary antibodies were from LI-COR Biosciences (Lincoln, NE, USA). Heregulinβ1 (HRG) was from Peprotech, Rocky Hill, NJ, USA.

Samples were prepared by adding RIPA lysis buffer and protease inhibitor (1:100; P2714; Sigma-Aldrich) and protein concentration was determined using a BCA protein assay. Protein was loaded and run on SDS-polyacrylamide gels before being transferred onto nitrocellulose membranes. The membranes were probed with mouse anti-aPKC-ι (1:1,000; 610207; BD Biosciences), rabbit anti-aPKC-ζ (1:2,000; HPA021851; Atlas Antibodies), mouse anti-total aPKC (1:1,000; sc-17781; Santa Cruz Biotechnologies), mouse anti-AKT(pan) (1:2,000; #2920; Cell Signaling Technology), rabbit anti-phospho Ser473 AKT (1:2,000; #4060; Cell Signaling Technology), rabbit-anti-Gasdermin-E (1:10,000; ab215191; Abcam), and mouse or rabbit anti-β-actin (1:10,000 [both antibodies]; #8457; Cell Signaling Technology or #3700; Cell Signaling Technology) and fluorescent secondary antibodies. Secondary antibodies included Alexa Fluor donkey anti-mouse 680 (1:10,000; A28183; Invitrogen) and Alexa Fluor donkey anti-rabbit 800 (1:10,000; A21039; Invitrogen). Total protein quantification was performed by staining membranes with Fast-Green FCF (74 (link)). All blots were scanned on a Licor Odyssey scanner and quantitation was performed using the Licor Imaging Software with target protein band intensity normalized to total protein.