Hrp coupled sheep anti mouse igg

Recombinant human TNF-α (PeproTech, Rocky Hill, NJ), ultrapure LPS and hygromycin B (InvivoGen, San Diego, CA), 10 mm ATP solution (Life Technologies), and [γ-³²P]ATP (SRP-501, Hartman Analytic, Germany) were from the indicated sources. Antibodies against caspase-8 (C-20), HA tag (Y-11), IKKα/β (H-470), NEMO/IKKγ (FL-419), and TAK1 (M-579) were from Santa Cruz Biotechnology. Mouse monoclonal antibodies for FADD (1/FADD) and RIP1 (38/RIP) were obtained from BD Biosciences. Antibodies specific for Atg3, calnexin (C5C9), GAPDH (14C10), MEKK3 (D36G5), pIκBα (14D4), and pIKKα/β (C84E11) were from Cell Signaling Technology. Anti-FLAG (M2), anti-HOIP (SAB2701544), and anti-CYLD (SAB4200061) were from Sigma-Aldrich. Anti-cFLIP (NF6) was from Enzo Life Sciences, and anti-cFLIP (G11) was from Santa Cruz Biotechnology. Antibodies recognizing HOIL-1 and SHARPIN were generously provided by Prof. Henning Walczak (University College London Cancer Institute), and human tumor cells lines were from Dr. Pablo Rodriguez-Viciana (University College London Cancer Institute). HRP-coupled sheep anti-mouse IgG (GE Healthcare), rabbit anti-goat IgG (Dako), and Fc fragment-specific goat anti-rabbit and light chain-specific mouse anti-rabbit IgG (both from Jackson ImmunoResearch Laboratories) were obtained from the indicated sources.

For Western-blotting, cell lysates were obtained by treating the cell monolayer with RIPA buffer complemented with protease and phosphatase inhibitors. Protein lysates were then cleared by centrifugation (17’000g for 20min). Total protein concentration was determined using bicinchoninic acid protein assay (Interchim, UP40840) and 25μg of denatured and reduced proteins of each sample was loaded on a 6% SDS-PAGE. For the IP3R1 immunoblotting (1/500, Abcam, ab5804) (S1B Fig), a 10% SDS-PAGE gel was used and the protein transfer was performed at low intensity overnight in a cold room. For the IP3R3 immunoblotting (1/1000, BD Biosciences, 610312) (S1C Fig), a 10% SDS-PAGE gel was used and the protein transfer was performed at low intensity overnight in a cold room.
After SDS-PAGE migration and electroblotting on polyvinylidene fluoride, the membranes were blocked with 5% non-fat milk and then incubated with the specific primary antibodies [rabbit anti-IP3R1 (Santa-Cruz, sc-28614; 1/500) and rabbit anti-TUBULIN (Santa-Cruz, sc-5286; 1/500)] (S1A Fig).
Blots were incubated with horseradish peroxidase (HRP)-coupled sheep anti-mouse IgG (GE Healthcare, NA931VS; 1/10000) and (HRP)-coupled goat anti-rabbit IgG (GE Healthcare, NA934VS; 1/10000), and developed with Clarity Western ECL Substrate (BioRad, 1705060). The band intensity was determined using Image Lab software (Bio-Rad).