Mouse anti actin

After washing with PBS, 3 × 10⁶ cells were directly lysed in pre-heated H8 buffer (20 mM Tris/HCl pH 7.5, 2 mM EGTA, 2 mM EDTA, 1% SDS, supplemented with 50 mM DTT) and boiled and homogenized at 95 °C in the presence of 4× Lämmli buffer. Proteins were separated on 12.5% or 15% denaturing SDS-PAGE gels and transferred to polyvinilydene difluoride (PVDF) membrane (Immobilon-FL, 0.45 μM, Merck Millipore, Zug, CH). After blocking, the membranes were probed overnight with the following primary antibodies: mouse anti-BCL-2 (clone 10C4, BioLegend); rat anti-BIM (clone 3C5) and rat anti-MCL-1 (clone 14C11), kind gifts from D. Huang (Parkville, Australia); mouse anti-GSK-3α/β (clone 0011-A/ 1H8) form Santa Cruz Biotechnology (Dellas, TX, US); rabbit anti-phospho-AKT (Ser473) (clone D9E) and mouse anti-AKT (clone 40D4) from Cell Signaling Technology (Danvers, MA, US); mouse anti-tubulin (clone B-5-1-2) from Sigma; mouse anti-GAPDH from Merck Millipore (Zug, CH); mouse anti-actin from BD Biosciences (San Jose, CA, US). For all immunoblots with total lysates, near-infrared fluorophore-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, DE) were used. All immunoblots were analyzed and quantified with the Odyssey^® Fc Dual-Mode Imaging System using the ImageStudio software 3.1.4 (LI-COR).

Antibodies: mouse anti-Cx43 IF1 (that binds aa 360–382, from Paul Lampe lab, see [55 (link)]), mouse anti-Cx43 252–270 (m252-270, Cat. # 610061; BD biosciences), mouse anti-actin (Cat. #MA1-744; Thermo scientific, Rockford, IL), rabbit anti-GST (Cat. # G7781; Sigma-Aldrich, St. Louis, MO). Pierce protein A agarose (Cat. # 20333; Thermo Scientific, Rockford, IL) beads were washed with 1% Bovine Serum Albumin (BSA) in 1X PBS and 10% SDS. The agarose beads were then washed three-four times in 1X PBS to remove the SDS and binding solution was added (1% BSA in 1X PBS). 4 μl of the antibody was added to the agarose beads. The antibody was allowed to bind to the resin at 4°C for 4 hours. After incubation, the agarose beads were washed four times with cold 1X PBS. More binding solution and 50 μg (for the overloading condition) or 20 μg (for the standard condition) of bait protein (CT fragments or deletions or F-actin) and prey protein (tubulin, GST-ZO-1^PDZ2, Drb^1-300, and transferrin) were added to the agarose beads. After overnight incubation, the agarose beads were washed four times with cold 1X PBS and 20 μl of 5% BME Novex Tricine loading sample buffer (Life Technologies, Inc., Carlsbad, CA) was added to each sample.

Neutrophils were lyzed with Pierce RIPA Buffer (Thermo Fisher Scientific) following the manufacturer’s instruction. Protease Inhibitor Cocktail (Sigma-Aldrich) was added to prevent protein degradation. 20 μg of the lysate was loaded on a 4–20% Tris-Glycine gel (Invitrogen) and ran in Novex Mini Cell (Invitrogen) following the manufacturer’s instructions. Human recombinant IL-26 (10 ng) was also loaded for control. Proteins were transferred on Amersham Hybond polyvinylidene difluoride membrane (GE Healthcare) and blocked for 1 h at room temperature with Tris-buffered saline with 0.1% Tween 20, 5% milk. The membrane was probed with primary antibodies (mouse anti-IL-26, clone 84, 1:1,000; or mouse anti-actin, BD, 1:5,000) and incubated overnight at 4°C. After washing, the membrane was probed with the corresponding secondary antibody HRP conjugated (rabbit anti-mouse HRP, 1:10,000; Dako) and incubated 1 h at room temperature. After washing, the membrane was revealed with WesternBright Sirius (Advasta) using Fusion Fx (Vilber Lourmat).