Ab82419

A549 cells and H1299 cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA were cultured for 2 days for protein isolation. In brief, cells were washed with PBS buffer and harvested with lysis buffer (100 mM Tris-HCl, pH = 7.4 l 0.15 M NaCl; 5 mM EDTA, pH = 8.0; 1% Triton X100; 5 mM DTT; 0.1 mM PMSF) to extract total proteins which were quantified with BCA Protein Assay Kit (Pierce, Rockford, IL, USA). To perform western blot analysis, 20 μg protein samples were mixed with loading buffer. Then SDS-PAGE electrophoresis and subsequent PVDF transmembrane were performed (Amersham Biosciences, Pollards Wood, UK). Membrane was blocked with 5% milk dissolved in TBST buffer for 1 h and then incubated with primary antibodies overnight at 4 °C. Primary antibodies used here were as follows: Rabbit anti-GMDS, Novus Biological, NBP1–33424 (1:500); Rabbit anti-CDKN1A, Abcam, ab7960 (1:500); Mouse anti-DDIT3, Abcam, ab11419 (1:1000); Rabbit anti-FAS, Abcam, ab82419 (1:1000); Rabbit anti-JUN, Abcam, ab32137 (1:1000); Rabbit anti-VEGFA, Abcam, ab183100 (1:500); mouse anti-Flag, Sigma, F1804 (1:1000); mouse anti-GAPDH, Santa-Cruz, sc-32,233 (1:2000). After washing with TBST buffer for three times, specific HRP conjugated secondary antibodies from Santa Cruz were added and immunoactivity was detected with ECL-Plus kit (Amersham Biosciences, Pollards Wood, UK).

Immunofluorescence studies of Fas and IFN-γ skins or HFs were performed using anti-Fas (ab82419, Abcam) and anti-IFN-γ (sc-373727, Santa Cruz Biotech) primary antibodies and corresponding anti-mouse/rabbit secondary antibodies. Nuclei were counterstained with DAPI (Invitrogen). Fluorescence was observed using a FluoView™ FV1000 confocal microscope (Olympus) and images were processed with Photoshop software. Images were post processed using Adobe Photoshop (rotate, crop, brightness, and contrast adjustments only).

Retinas were homogenized in lysis buffer (20 mM Tris [pH 8.0], 135 mM NaCl, 1% sodium dodecyl sulfate [SDS], and 10% glycerol supplemented with protease inhibitors) and centrifuged at 14,000 rpm for 5 min. The supernatants were collected and diluted in Laemmli sample buffer 5× (4% SDS, 10% glycerol, 0.004% bromophenol blue, 0.1 M dithiothreitol, and 0.125 M Tris, pH 6.8). Extracts were processed using standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting procedures using precast Mini-PROTEAN TGX gels (Bio-Rad). Equal volumes were loaded on the gels. Antibodies include anti-acetyl p53 K382 (2570, Cell Signaling), anti-acetyl p53 K381 (ab61241, abcam), anti-c-Jun (9165, Cell Signaling), anti-Erk1/2 (9102, Cell Signaling), anti-Fas (ab82419, abcam), anti-JNK (9252, Cell Signaling), anti-p21 (sc-397, Santa Cruz), anti-p53 (MAB1746, R&D systems), anti-Phospho-Erk1/2 T202/Y204 (9255, Cell Signaling), anti-Phospho-JNK T183/Y185 (9255, Cell Signaling), anti-Phospho-c-Jun s63 (2301, Cell Signaling), anti-Phospho-p53 Ser15 (9286, Cell Signaling), anti-PUMA (7467, Cell Signaling), and anti-α-tubulin (T5168, Sigma). Secondary antibodies were obtained from Southern Biotech and Jackson ImmunoResearch. Quantity One software (Bio-Rad) was used for quantification.

The preparation of DHP1808 was as per our previous reports [38 (link)]. VX-765 and Z-VAD-FMK were obtained from Selleckchem Co. Ltd. (Shanghai, China). The antibodies recognizing FADD (14906-1-AP), Bcl-2 (12789-1-AP), Bax (50599-1-AP), Cytochrome C (10993-1-AP), β-Catenin (51067-2-AP), CDK2 (10122-1-AP), CDK4 (11026-1-AP), CDK6 (14052-1-AP), CyclinB1 (55004-1-AP), p21 (10355-1-AP), E-Cadherin (20874-1-AP), MMP2 (10373-1-AP), MMP9 (10375-1-AP), ZEB1 (21544-1-AP), GAPDH (60004-1-Ig) and β-actin (60008-1-Ig), MNK1 (10136-1-AP), MDM2 (19058-1-AP), and p53 (10442-1-AP) were purchased from Proteintech (Wuhan, China). The antibody recognizing Fas (Ab82419), FasL (Ab68338), Caspase-3 (Ab13847), PARP (Ab32138), N-Cadherin (Ab76011), Met (Ab51067), Phos-Met (Ab68141), ERK1/2 (Ab17942), Phos-ERK1/2 (Ab76299), JNK (Ab179461), Phos-JNK (Ab124956), phos-MNK1 (ab109102), Caspase-1 (ab179515), and GSDMD (ab215203) were purchased from Abcam (Cambridge, MA, USA). The antibody recognizing Bad (9239), Bim (2933), Caspase-8 (9746), Caspase-9 (9508), p27 (3686), Cdc37 (10218-1-AP), cRaf (2330), Phos-cRaf (2330), BRaf (2330), Phos-bRaf (2330), c-Myc (5605), p90RSK (9326), Phos-p90RSK (9326), Hsp90 (4877), EGFR (2232), Phos-EGFR (4407), Akt1 (4691), phos-Akt308 (4056), and phos-Akt473 (4060) were purchased from Cell Signaling Technology (Danvers, MA, USA).

The spleen was fixed in 10 % neutral formaldehyde and embedded in paraffin wax. The 4-μm-thick sections were dewaxed in xylene and rehydrated by taking through a graded series of ethanol. The endogenous peroxidase activity was blocked with 3 % hydrogen peroxide for 15 min. Sections were incubated with rabbit anti-Fas antibody (ab82419, Abcam Ltd, Hong Kong, 1:100 dilution) or rabbit anti-FasL antibody (sc-834, Santa Cruz Biotechnology, USA, 1:200 dilution) overnight at 4°C and then incubated with goat polyclonal secondary antibody to rabbit IgG-H&L-HRP (ab6721, Abcam Ltd, Hong Kong, 1:500 dilution) for 30 min at 37 °C. Negative controls were incubated with normal rabbit serum instead of the first antibody. Finally, sections were stained by using a 3,3'-diaminobenzidine (DAB) kit (Maixin Biotechnology Company, China) and counterstained with hematoxylin, dehydrated through a graded series of ethanol solutions, cleared with xylene, and mounted with coverslips. The analysis was performed using a scoring system as described previously [35 (link)]. All the slides were scored by two pathologists who were blinded to the pathology and experimental protocol.