Granzyme b

Cells were lysed on ice in freshly prepared RIPA lysis buffer [150 mM NaCl, 1.0% IGEPAL^® CA‐630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, supplemented with Complete protease inhibitor cocktail without EDTA (Roche)]. Samples were clarified by centrifugation at 20,000 relative centrifugal force (r.c.f.) for 20 min at 4°C, and protein concentrations were determined using a BCA protein assay. Twenty micrograms of protein lysates was used for immunoblotting, with the proteins resolved on 4–12% NuPAGE Bis‐Tris Gels (Invitrogen) run with 1× MOPS buffer (Invitrogen) for 45 min at 200 V. Proteins were transferred onto PVDF membranes using a wet‐blotting system for 1 h at 400 mA. PVDF was blocked overnight in 5% skim milk powder in PBS‐T and then probed with primary and then secondary antibodies in 1% skim milk in PBS‐T, with three washes of PBS‐T between probes. Primary antibodies against PARP (# 9542, Cell Signaling), TACC3 Antibody (D‐2, sc‐48368, Santa Cruz), CDC‐42 (P1, SC‐87, Santa Cruz), cytochrome C (7H8.2C12, Abcam), NCAPH (HPA002647, Sigma‐Aldrich), and granzyme B (# 4275, Cell Signaling) were utilized at 1:5,000. Blots were developed with HRP‐conjugated secondary antibody diluted 1:10,000 in PBS‐T using chemiluminescence method (ECL) using either Hyperfilm detection (GE Healthcare Life Sciences) or digital detection using a Gel Doc™ XR+ System (Bio‐Rad).

For IHC staining of the xenografts, tumor tissues were fixed, embedded, and sectioned (3 μm thick). Immunohistochemistry staining for human and mouse tissues was performed in accordance with standard procedures [27 (link)]. The following antibodies were used: primary antibody CD3+ (dilution 1:200) or Granzyme B (dilution 1:400) (Cell Signaling Technology, USA) for mouse tissues, and anti-PD-L1 (dilution 1:1000) (E1L3N, Cell Signaling Technology) or anti-p-STAT3 (dilution 1:500) (D3A7, Cell Signaling Technology) for human tissues.

Stimulated CD8⁺ T cells were lysed in 1× RIPA buffer and proteins extracted by sonication.
Protein concentration was determined using the Pierce™ BCA™ Protein Assay Kit (Thermo Fisher). Equivalent amounts of proteins were denatured in Laemmli buffer under reducing conditions, separated by 7%–10% SDS‐PAGE and transferred to PVDF‐membrane using a transblot electrophoresis transfer cell (Fisherbrand). Membranes were blocked with 5% skim milk powder in Tris–HCl‐buffered saline/0.1% Tween (TBST) for 1 h, incubated overnight with primary antibodies against perforin (Invitrogen), granzyme B (Cell Signaling), and GAPDH (Cell Signaling), washed in TBST and incubated with horseradish peroxidase‐coupled secondary antibodies (GE Healthcare) for 1 h. For protein detection, an enhanced chemiluminescence detection reagent (BioRad) was used. Densitometric quantification was done with Quantity one software (Bio‐Rad).