Ab202068

Melanoma A375 cells were harvested and homogenized in lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The primary antibodies used were p75^NTR (#ab52987; Abcam, Cambridge, UK), NF‐κB p65 (#8242; CST, Danvers, MA, USA), IκBα (#9242; CST, Danvers, MA, USA), Phospho‐IκBα (#2859; CST, Danvers, MA, USA), caspase‐3 (ab214430; Abcam, Cambridge, UK), caspase‐9 (ab202068; Abcam, Cambridge, UK), Bax (ab32503; Abcam, Cambridge, UK), Bcl‐2 (ab32124; Abcam, Cambridge, UK), Lamin B1 (Beyotime, Shanghai, China), inhibitor of apoptosis protein 2 (c‐IAP2; #380798; ZEN BIO, Chengdu, China), bFGF (#381676; ZEN BIO, Chengdu, China) and β‐actin (Sino Biological, Beijing, China). β‐actin or Lamin B1 expression served as an internal control. For immunofluorescence analysis, cells were incubated with anti‐(human NF‐κB p65) IgG (1 : 100). The primary antibody was detected using a Cy3‐labeled IgG (1 : 100; Beyotime, Shanghai, China), and nuclei were stained with 4',6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). Microscopic analysis was performed using a Nikon E600 microscope (Tokyo, Japan).

The tissue proteins were extracted with RIPA lysis buffer. After being equalized for total protein concentration, the protein was separated by SDS-PAGE and subjected to semidry blotting onto nitrocellulose membranes. The membrane was blocked and incubated overnight with rabbit anti-mouse caspase-3, caspase-9, Bax, Bcl-2, and GAPDH antibodies (ab32503, ab182858, ab184787, and ab202068, Abcam; 5174, Cell Signaling Technology) at 4°C. After being incubated with the peroxidase-conjugated goat anti-rabbit IgG (7074, Cell Signaling Technology), the blot was visualized by ECL™ (P0018A, Beyotime Technology) and X-ray film. Then, the expression of apoptosis-associated proteins is shown relative to that of GAPDH using Quantity One software.

Lysis of cells was performed using lysis buffer (200 μL/well). Constituents of lysis buffer were NaCl (150 mM), Tris (pH 7.6, 50 mM), Triton X-100 (1%), including phosphatase and protease inhibitors. Extracted proteins (40 μg of the total proteins) were separated by performing SDS–PAGE (7.5%). Procedure of western blotting was carried out as described by Waraich et al. and Run et al. [26 (link),27 (link)]. Following antibodies were used for western blotting: anti-GFAP, Abcam (ab7260); anti-Oligo2, Abcam (ab136253); anti-Iba1, Abcam (ab5076); anti-beta actin, Abcam (ab115777); anti-caspase-3, Abcam (ab13847); anti-cleave caspase-3, Abcam (ab32042); anti-caspase-9, Abcam, (ab202068); anti-cleave caspase-9, Abcam (ab25758); anti-tau, Abcam (ab76128); anti-tau phospho, Abcam (ab109390); anti-CaMKII, Abcam (ab22609); and anti-CaMKII phospho, Abcam (ab171095). Samples of protein were transferred to nitrocellulose membrane post-electrophoresis. Membrane was blocked by bovine serum albumin or skim milk for 2 h and later incubated overnight with primary antibody (Ab) at 4°C. After washing, membrane was incubated with secondary-Ab (anti-rabbit/mouse Ig-G conjugated to horseradish peroxidase) for 1 h at room temperature. Enhanced chemiluminescence was carried out to visualize protein expression. All western blot experiments were in triplicate. ImageJ was used for protein quantification.

PD-1 downstream signaling molecules in CD4⁺ and CD8⁺ T cells were measured by Western blot analysis at 96 hpi. The primary antibodies were PI3K (ab227204, 1:1000, Abcam), p-PI3K (ab182651, 1:1000, Abcam), AKT (#9272, 1:1000, Cell Signaling Technology, USA), p-AKT (Ser473) (#9271, 1:1000, Cell Signaling Technology), caspase 9 (ab202068, 1:1000, Abcam), caspase 3 (19677-1-AP, 1:500, Proteintech), ERK (#4695S, 1:1000, Cell Signaling Technology), p-ERK (Thr202/Tyr204) (#9101, 1:1000, Cell Signaling Technology), mTOR (ab2732, 1:1000, Abcam), p-mTOR (ab84400, 1:1000, Abcam), and β-actin (60008-1-lg, 1:15000, Proteintech), which was used as an internal control. The secondary antibodies were HRP-conjugated affinipure goat anti-mouse IgG (H+L) (SA00001-1, 1:8000, Proteintech) and HRP-conjugated affinipure goat anti-rabbit IgG (H+L) (SA00001-2, 1:8000, Proteintech).

OS cells were fixed using 4% paraformaldehyde (PFA) for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. After blocked with blocking buffer (P0260, Beyotime, China) for 10 min at room temperature, the slices were incubated with antibodies against cleaved-caspase-3 (ab13847, Abcam, USA), cleaved-caspase-9 (ab202068, Abcam, USA) or LC3B (ab48394, Abcam, USA) overnight at 4 °C. The slices were imaged using confocal microscopy.