Anti granzyme b

Cells were lysed in buffer containing 0.5% Nonidet P-40, 20 mM Tris-HCl (pH=7.4), 150 mM NaCl, 2 mM sodium orthovanadate, 10% glycerol, and complete protease inhibitor (Roche Diagnostics, Indianapolis, IN). Protein concentration was determined by Bradford assay (Bio-rad, Hercules, CA). Protein lysates were separated by SDS-PAGE using 12.5% acrylamide gels and transferred to Immobilon-FL (Millipore, Billerica, MA) membranes. Membranes were blocked with 4% w/v milk in PBS and then incubated with primary antibody in PBS containing 4% milk and 0.1% v/v Tween-20 overnight at 4°C. After washing with PBS containing 0.1% Tween-20, membranes were incubated with fluorescent-conjugated species-specific secondary antibodies in PBS containing 4% milk, 0.1% Tween-20, and 0.01% SDS. Images were acquired with an Odyssey CLx using Image Studio software (LI-COR, Lincoln, NE). The fluorescence signals for each sample were background subtracted and normalized to α-tubulin using Image Studio software. The following antibodies were used for immunoblot analysis: anti-Fasligand (FasL) (R&D Systems, MAB5262), anti-α-tubulin (Cell Signaling Technology), anti-granzyme B (Cell Signaling Technology), IRDye 800CW goat anti-rat (LI-COR), IRDye 680RD goat anti-mouse IgG1 (LI-COR), IRDye 800CW goat anti-rabbit (LI-COR).

For ex vivo analysis, mice were sacrificed on the specified day and the entire tumor excised and either lysed for Western blot analysis or fixed in 10% formalin and paraffin embedded for immunohistochemical or immunofluorescence staining. For Western blot analyses, anti-CD8 (Ab108292, Abcam, Cambridge, MA), anti-CD3 (sc-20047, Santa Cruz Biotechnology, Dallas, TX, anti-CD4 (sc-19643, Santa Cruz), anti-FoxP3 (12653s, Cell Signaling Technologies, Danvers, MA), anti-pSTAT (700349, ThermoFisher, Waltham, MA), anti-Granzyme B (4275s, Cell Signaling Technologies) and anti-β-actin (Cell Signaling Technologies 4970S) were used at the manufacturer’s recommended concentration. For immunohistochemistry analysis, granzyme B (Ab4059, Abcam) and CD3 (Ab56090, Abcam) antibodies were detected using biotinylated goat-anti-rabbit antibodies with the Signal Stain Boost IHC Detection Reagent (Abcam). For immunofluorescence staining, granzyme B was detected with Ab4059 and AlexaFluor 488 goat anti-rabbit secondary (Life Technologies); CD3 was detected with anti-CD3 clone PC3/188A (Santa Cruz) and AlexaFluor 594 goat anti-mouse secondary (Life Technologies).