Rabbit monoclonal antibodies

The anti-HER3 antibody 9F7-F11 has been described elsewhere [2 (link), 32 (link), 33 (link), 36 (link)]. Rabbit monoclonal antibodies against HER3 and phosphorylated (p) HER3 (Tyr1289), PARP (clone 46D11), cleaved caspase-8 (Asp391; clone 18C8), caspase-9 and cleaved caspase-9, caspase-3 (clone 8G10) and cleaved caspase-3, XIAP, DR5 (clone D4E7), BIM, c-FLIP (clone D16A8), USP9X, β-actin and β-tubulin were from Cell Signaling Technology (Danvers, MA). The mouse monoclonal antibody against ITCH was from BD Biosciences (San José, CA; ref.611199). The rabbit polyclonal antibodies against HER3 (clone C17), USP8, USP9X, c-FLIP_L/S (clone H-202), BAX, FAS, TRAIL, DcR2, cIAP2, and BID were from Santa Cruz Biotechnology (Santa Cruz, CA). For detection of activated ITCH, a rabbit anti-pITCH (Thr222) antibody from Millipore (Billerica, MA) was used. The human recombinant NRG1-β1 extracellular domain (ECD) was from RD Systems (Minneapolis, MN) and was used at 100 ng/ml. The proteasome inhibitor MG132 and chlorimipramine (CI) were from Sigma-Aldrich (St Louis, MO). The control IgG antibody used for co-immunoprecipitation experiments was from Santa Cruz Biotechnology.

Western blot was performed as described in [12 (link)], using rabbit monoclonal antibodies (Cell Signaling, Danvers, MA, USA) against cyclin B1 (1:500), Aurora kinase A (1:500), topoisomerase IIα (1:500), or calnexin (1:1000) and peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:5000; Calbiochem, San Diego, CA, USA).

Syngeneic tumor tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at a 4 μm thickness on a Leica RM2235 microtome. Sections were stained with rabbit monoclonal antibodies (Cell Signaling Technology) against murine CD8α (clone D4W2Z) and PD-L1 (clone D5V3B) using the automated Leica BOND Rx platform. Stained slides were imaged with a Leica Aperio VERSA scanner and analyzed with HALO software (Indica Labs). The percentage positivity for each marker was calculated as the density of positive cells per mm² divided by the total number of cells per mm².

Western blot analysis was used to detect protein expression in breast cancer cells. Cells were lysed in a standard buffer (0.1% (w/v) Sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate, 1% Triton X-100, 1 μM Ethylenediaminetetraacetic acid, 2% (v/v) protease inhibitor cocktail, 10 µM of sodium fluoride and 2% (v/v) phosphatase inhibitor cocktail). Protein concentration was determined using BCA assay (Pierce, USA). Total protein lysate (60μg) was resolved by SDS-PAGE (BioRAD, United Kingdom), immunoblotted with antibodies according to manufacturer’s instructions, detected using rabbit monoclonal antibodies (all at 1:1000 dilution, cell Signalling Technology, USA) and immuno-complexes were visualised by enhanced chemiluminescence (Amersham, UK) on a Syngene GeneGnome imaging system. The intensity of the bands was quantified using GeneSnap software (Syngene, UK) and level of actin was used for normalization.

Western blot analysis was used to detect Cnr1 and FABP4 expression in cultured mouse preadipocyte 3T3‐L1 cells as previously described in Sophocleous et al. (2015). Protein concentration was determined using BCA assay (Pierce, USA). Total protein (70 μg) was resolved by SDS–PAGE, transferred onto PVDF membranes (Bio‐RAD, Hamburg, Germany) and immunoblotted with antibodies according to manufacturer's instructions. Detection of human FABP4 (Cell Signalling Technology, Danvers, Massachusetts, USA) and mouse Cnr1 (Cayman Chemical, Hamburg, Germany) was performed using rabbit monoclonal antibodies (Cell Signalling Technology) followed by appropriate secondary antibody coupled to horseradish peroxidase and then visualized using chemiluminescence (Amersham, Hamburg, Germany) on a Syngene GeneGnome imaging system. The intensity of the bands was quantified using GeneSnap software (Syngene, UK), and level of actin expression was used for normalization.