Odyssey classic imaging system

Proteins were transferred to polyvinylidene fluoride membranes after SDS–PAGE using a Bio-Rad trans-blot turbo system. Membranes were blocked with 5% nonfat dry milk in TBS and 0.1% Tween for 1 h prior to incubation with appropriate caspase-6 antibodies (Cell Signaling, catalog nos. 9761 and 9762). Primary antibodies were utilized at dilutions of 1:1000. Secondary antibodies obtained from LiCor were used at dilutions of 1:20000 (IRDye IgG antibodies, catalog nos. 5470S, 5151S, 5257S, and 5366S). Images were both collected and analyzed using a LiCor Odyssey classic imaging system.

The cells were collected and washed once with ice-cold PBS and resuspended in modified RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 0.1% SDS, 1 x Halt protease and phosphatase inhibitor cocktail, 1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 10 μg/ml aprotinin, for 10 minutes. The lysates were further sonicated in ice-cold water for 4 minutes and then centrifuged at 16,000×gfor 15 minutes at 4°C. Supernatant was collected and protein concentration was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific) or Bradford reagent (Bio-Rad). Equal amount of sample was resuspended in lx SDS loading dye, boiled at 100°C for 5 minutes, loaded on a 10, 12.5 or 15% SDS-PAGE gel, and resolved electrophoretically at 100V for 1.5 h. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 100V for 2 h. Non-specific binding sites on the membrane were blocked by incubating the membrane in 5% BSA solution for 1 h at room temperature. The membranes were probed with the indicated primary antibody (1: 1000), washed three times for 5 minutes each, followed by incubation with a secondary fluorescence labeled antibody (1:7500). The antibody-bound proteins were detected by LI-COR Odyssey classic imaging system. Band intensity was analyzed using ImageJ software.

Crushed C lectularius salivary gland was mixed with diluted 4× loading buffer (LI-COR), denatured, and separated by SDS-PAGE on 4% to 15% Mini-PROTEAN TGX gels (Bio-Rad) with diluted 10× Tris/glycine/SDS buffer (Bio-Rad) at 5 μg of protein per well. The separated proteins were transferred to PVDF transfer membranes (Thermo Scientific) through a wet transfer process with transfer buffer (20% methanol, 10% Tris-glycine). The membranes were then blocked with Odyssey Blocking Buffer (LI-COR) for 1 hour and incubated overnight with participant serum diluted 1:10 in TBST (Tris-buffered saline [Bio-Rad] plus 0.1% Tween 20 [Fisher Scientific]). Fluorescence-based Western blotting was then performed on the membranes by probing bands with an IRDye 800CW–labeled goat antihuman IgG secondary antibody (LI-COR) diluted 1:7,000 in TBST. The membranes were then dry scanned on an Odyssey Classic Imaging system (LI-COR).