Sc 17781

For sample preparation, when the cell culture reached 80% confluence, the cells were collected and split for 40 minutes on ice, the supernatant was denatured in 5x loading buffer containing SDS in boiling water for 10 minutes. We suggest not to boil the sample after lysis when preparing protein samples for ABCB1 determination by western blot. Next, 5% bovine serum albumin (BSA) dissolved in TBS containing 0.1% Tween-20 (TBST) was used for blocking and antibody dilution. The following antibodies and dilutions were used: actin (1:1,000, HC201, TransGen), CCL20 (1:1,000, AF360, R&D), p65 (1:1,000, 8242S), p-p65 (1:1,000, 3033S), p38 (1:1,000, 9212), p-p38 (1:1,000, 4631), p-PKCζ (1:1,000, 9378), ABCB1 (1:1,000, 13342S) (all from Cell Signaling Technology, USA), PKCζ (1:100, sc-17781, Santa Cruz), goat anti-mouse IgG-HRP (1:5,000, sc-2005, Santa Cruz), goat anti-rabbit IgG (1:5,000, sc-2004, Santa Cruz), donkey anti-goat IgG-HRP (1:2,000, SA00001-3, Proteintech). Western HRP Substrate (WBLUF0500, Millipore) was used to detect horseradish peroxidase–conjugated secondary antibodies.

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.